Identification of resistance genes from wild relatives of banana and their uses in controlling panama disease

ABSTRACT

The present disclosure provides compositions and methods for providing broad-based resistance to fungal pathogens, such as a  Fusarium  fungi, and plants derived therefrom.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/866,872, filed on Jun. 26, 2019, and of U.S.Provisional Patent Application No. 62/912,010, filed on Oct. 7, 2019,the entire contents of each of which are herein incorporated byreference.

FIELD

The present disclosure generally relates to the field of agriculturalindustry, especially production of consumer crops with pathogenicresistance. More particularly, the present disclosure relates tocompositions and methods for generating plants that possess traitsresistant to fungal pathogens such as the soil-born Fusarium fungiand/or that show resistance to diseases caused by said fungal pathogens.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy. The contents of the text filesubmitted electronically herewith are incorporated herein by referencein their entirety: A computer readable format copy of the SequenceListing (filename: EVOL_009_02US_SubSeqList_ST25.txt, date recorded:Jul. 22, 2020; file size: 27.0 kilobytes).

BACKGROUND OF THE DISCLOSURE

Bananas are one of the world's biggest fruit crops, totaling over 100million metric tons. Bananas are the most popular fruit in developedcountries, and are an important food and income source for a largepercentage of the world, providing food security in many tropical andsubtropical nations. In fact, bananas are the fourth most important foodcrop in developing nations where the vast majority of bananas areproduced and consumed locally. The major producing countries are India,China, Ecuador, Brazil, and some African countries.

About 15 percent of banana production is traded on the global market,generating about $8 Billion annually. The top exporting countries areEcuador, Philippines, Costa Rica, and Columbia.

However, this important crop is now severely threatened by FusariumWilt, also known as Panama Disease, caused by the fungus Fusariumoxysporum f sp. cubense (Foc).

Half of the commercial banana crop world-wide and even up to 90% ofbanana exports in some countries consist of a single group of cultivars,the Cavendish genotypes, which are propagated clonally. Also, most ofthe commercially traded bananas and many of the locally consumed bananasare clonally cultivated with a single crop in a given area, known as‘monoculture.’ The monoculture has been widely practiced by farmers tomass-produce highly demanded crops such as banana, which is easilyaffected by a range of fungal, viral, bacterial and nematode diseases.Clearly, the current expansion of the Panama disease epidemic isparticularly destructive due to the massive monoculture of susceptibleCavendish bananas.

Cavendish bananas are the fruits of one of a number of banana cultivarsbelonging to the Cavendish subgroup of the AAA banana cultivar group.The same term is also used to describe the plants on which the bananasgrow. They include commercially important cultivars like ‘DwarfCavendish’ (1888) and ‘Grand Nain’ (the “Chiquita banana”). ‘Williams’is a cultivar of the ‘Giant Cavendish’ type in the Cavendish subgroup.It is one of the most widely grown cultivars in commercial plantations.‘Formosana’ is another name for the somaclonal variant ‘GCTCV-218,’which has some resistance to Fusarium wilt TR4. Other representativecommercial cultivars include ‘Masak Hijau’ and ‘Robusta.’ Since the1950s, these cultivars have been the most internationally tradedbananas. They replaced the Gros Michel banana (commonly known as Kampalabanana in Kenya and Bogoya in Uganda) after it was devastated by Panamadisease.

Thus, there is an urgent need in the art for bananas that are resistantto Fusarium Wilt or Panama Disease.

SUMMARY OF THE DISCLOSURE

The present disclosure solves the aforementioned Panama Disease problemby identifying the underlying genetic architecture giving rise toresistance. Furthermore, the disclosure teaches methodology by whichthis resistance genetic architecture can be imported into diseasesusceptible bananas and thus render these bananas disease resistant. Theimportation of this genetic architecture can take many forms, aselaborated upon herein, including: traditional plant breeding,transgenic genetic engineering, next generation plant breeding (CRISPR,base editing, MAS, etc.), and other methods.

In some embodiments as provided herein are isolated nucleic acidmolecules comprising nucleic acid sequence SEQ ID NO: 14 coding forsusceptibility to Fusarium oxysporum race 4 when expressed in a plant,wherein SEQ ID NO: 14 is modified by one, two, three or four nucleicacid substitutions so that the resulting nucleic acid sequence codes forresistance to Fusarium oxysporum race 4 when expressed in a plant. Insome embodiments, the isolated nucleic acid molecule includes nucleicacid substitutions comprising replacing a T corresponding to position148 of SEQ ID NO: 14 with a G (148T>G). In some embodiments, theisolated nucleic acid molecule includes nucleic acid substitutionscomprising replacing a T corresponding to position 323 of SEQ ID NO: 14with an A (323T>A). In some embodiments, the isolated nucleic acidmolecule includes nucleic acid substitutions comprising replacing a Gcorresponding to position 344 of SEQ ID NO: 14 with a C (344G>C). Insome embodiments, the isolated nucleic acid molecule includes nucleicacid substitutions comprising replacing an A corresponding to position347 of SEQ ID NO: 14 with a T (347A>T). In some embodiments, theisolated nucleic acid molecule includes nucleic acid substitutionscomprising replacing a T corresponding to position 323 with an A(323T>A), replacing a G corresponding to position 344 with a C (344G>C),and replacing an A corresponding to position 347 with a T (347A>T), andwherein all positions are based on SEQ ID NO: 14. In some embodimentsthe isolated nucleic acid molecule of SEQ ID NO: 14 codes for an aminoacid sequence of SEQ ID NO: 15 and wherein the nucleic acidsubstitutions result in replacing a Leucine corresponding to position 50of SEQ ID NO: 15 with a Valine (50L>V). In some embodiments, theisolated nucleic acid molecule includes SEQ ID NO: 14 which codes for anamino acid sequence of SEQ ID NO: 15 and wherein the nucleic acidsubstitutions result in replacing a Valine corresponding to position 108of SEQ ID NO: 15 with a Glutamic Acid (108V>E). In some embodiments, theisolated nucleic acid includes a SEQ ID NO: 14 which codes for an aminoacid sequence of SEQ ID NO: 15 and wherein the nucleic acidsubstitutions result in replacing an Arginine corresponding to position115 of SEQ ID NO: 15 with a Proline (115R>P). In some embodiments, theisolated nucleic acid molecule includes a SEQ ID NO: 14 which codes foran amino acid sequence of SEQ ID NO: 15 and wherein the nucleic acidsubstitutions result in replacing an Aspartic Acid corresponding toposition 116 of SEQ ID NO: 15 with a Valine (116D>V). In someembodiments, the isolated nucleic acid molecule includes a SEQ ID NO: 14which codes for an amino acid sequence of SEQ ID NO: 15 and wherein thenucleic acid substitutions result in replacing a Valine corresponding toposition 108 of SEQ ID NO: 15 with a Glutamic Acid (108V>E), an Argininecorresponding to position 115 of SEQ ID NO: 15 with a Proline (115R>P),and an Aspartic Acid corresponding to position 116 of SEQ ID NO: 15 witha Valine (116D>V).

In some embodiments, the expression occurs in a plant cell, planttissue, plant cell culture, plant tissue culture, or whole plant. Insome embodiments the expression occurs in a Musa cell, tissue, cellculture, tissue culture, or whole plant. In some embodiments, theexpression occurs in a Musa acuminata cell, tissue, cell culture, tissueculture or whole plant.

In some embodiments, a nucleic acid construct comprises the nucleic acidsequences of the present invention which are operably linked to apromoter capable of driving expression of the nucleic acid sequence. Insome embodiments, the promoter is a plant promoter. In some embodiments,the promoter is a 35S promoter. In some embodiments, the promoter iscoded by SEQ ID NO: 31.

In some embodiments, a transformation vector comprises the nucleic acidconstructs of the present invention.

In some embodiments, provided herein is a method of transforming a plantcell comprising introducing the transformation vectors of the presentinvention into a plant cell, whereby the transformed plant cellexpresses the nucleic acid sequence coding for resistance to Fusariumoxysporum race 4. In some embodiments, the method uses a plant cellwhich is aMusa plant cell. In some embodiments, the method uses a plantcell which is aMusa acuminata plant cell.

In some embodiments, the transformed plant tissue is produced from thetransformed plant cell. In some embodiments, a transformed plantlet isproduced from the transformed plant tissue. In some embodiments, a cloneis produced from the transformed plantlet. In some embodiments, themethod comprises growing the transformed plantlet or clone of thetransformed plantlet into a mature transformed plant. In someembodiments, the mature transformed plant is a Musa plant and the maturetransformed Musa plant is capable of producing fruit. In someembodiments, the methods of the present invention include furtherproducing clones of the mature transformed Musa plant. In someembodiments, the mature transformed Musa plant or clone of the maturetransformed Musa plant are used in breeding methods.

In some embodiments, the present invention provides an isolated aminoacid molecule comprising an amino acid sequence of SEQ ID NO: 15 codingfor a protein that when produced in a plant results in susceptibility toFusarium oxysporum race 4, wherein SEQ ID NO: 15 is modified by one,two, three or four amino acid substitutions so that it codes for aprotein which when produced in a plant results in resistance to Fusariumoxysporum race 4. In some embodiments, the amino acid substitutionscomprise replacing a Leucine corresponding to position 50 of SEQ ID NO:15 with a Valine (50L>V). In some embodiments, the amino acidsubstitutions comprise replacing a Valine corresponding to position 108of SEQ ID NO: 15 with a Glutamic Acid (108V>E). In some embodiments, theamino acid substitutions comprise replacing an Arginine corresponding toposition 115 of SEQ ID NO: 15 with a Proline (115R>P). In someembodiments, the amino acid substitutions comprise replacing an AsparticAcid corresponding to position 116 of SEQ ID NO: 15 with a Valine(116D>V). In some embodiments, the amino acid substitutions comprisereplacing a Valine corresponding to position 108 of SEQ ID NO: 15 with aGlutamic Acid (108V>E), an Arginine corresponding to position 115 of SEQID NO: 15 with a Proline (115R>P), and an Aspartic Acid corresponding toposition 116 of SEQ ID NO: 15 with a Valine (116D>V). In someembodiments, the protein production occurs in a plant cell, planttissue, plant cell culture, plant tissue culture, or whole plant. Insome embodiments, the protein production occurs in a Musa cell, tissue,cell culture, tissue culture, or whole plant. In some embodiments, theprotein production occurs in a Musa acuminata cell, tissue, cellculture, tissue culture or whole plant.

In some embodiments, the nucleic acid constructs of the presentinvention comprise a nucleic acid sequence coding for resistance toFusarium oxysporum race 4 when expressed in a plant, wherein saidnucleic acid sequence is selected from the group consisting of SEQ IDNO: 2, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 18, SEQ IDNO: 21, SEQ ID NO: 24 and SEQ ID NO: 29, and wherein the nucleic acidsequence is operably linked to a promoter capable of driving expressionof the nucleic acid sequence. In some embodiments, the promoter is aplant promoter. In some embodiments, the promoter is a 35S promoter. Insome embodiments, the promoter is coded by SEQ ID NO: 31. In someembodiments, a transformation vector comprises the nucleic acidconstructs of the present invention. In some embodiments, the presentinvention provides methods of transforming a plant cell comprisingintroducing the transformation vector into a plant cell, whereby thetransformed plant cell expresses the nucleic acid sequence coding forresistance to Fusarium oxysporum race 4. In some embodiments, the plantcell is a Musa plant cell. In some embodiments, the plant cell is a Musaacuminata plant cell. In some embodiments, the methods further compriseproducing transformed plant tissue from the transformed plant cell. Insome embodiments, a transformed plantlet is produced from thetransformed plant tissue. In some embodiments, the methods furthercomprise producing a clone of the transformed plantlet. In someembodiments, the methods further comprise growing the transformedplantlet or clone of the transformed plantlet into a mature transformedplant. In some embodiments, the mature transformed plant is a Musa plantand the mature transformed Musa plant is capable of producing fruit. Insome embodiments, the methods further comprise producing clones of themature transformed Musa plant. In some embodiments, the maturetransformed Musa plant or clone of the mature transformed Musa plant isused in a breeding method.

In some embodiments, the invention provides a banana breeding methodcomprising crossing a first Musa plant comprising a nucleic acidsequence coding for resistance to Fusarium oxysporum race 4 with asecond Musa plant that is susceptible to Fusarium oxysporum race 4 andselecting resultant progeny of the cross based on their resistance toFusarium oxysporum race 4, wherein said nucleic acid sequence coding forresistance to Fusarium oxysporum race 4 is selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24 and SEQ ID NO: 29. In someembodiments, the banana breeding methods further comprise producingclones of the resultant progeny of the cross wherein the clones areselected based on their resistance to Fusarium oxysporum race 4. In someembodiments, the first and second Musa plants are from different Musaspecies. In some embodiments, the first and second Musa plants are fromthe same Musa species. In some embodiments, the first and/or second Musaplant is a Musa acuminata plant. In some embodiments, the progeny of thecross that display resistance to Fusarium oxysporum race 4 are selectedusing molecular markers that are designed based on the nucleic acidsequence coding for resistance to Fusarium oxysporum race 4 that ispresent in the first Musa plant used in the cross.

In some embodiments, the present invention provides methods forobtaining a Musa acuminata plant cell with a silenced endogenous genecoding for susceptibility to Fusarium oxysporum race 4, the methodcomprising introducing a double-strand break to at least one site in anendogenous gene coded by SEQ ID NO: 14 to produce a Musa acuminata plantcell with a silenced endogenous gene coding for susceptibility toFusarium oxysporum race 4. In some embodiments, the methods furthercomprise generating a Musa acuminata plant from the Musa acuminata plantcell with a silenced endogenous gene coding for susceptibility toFusarium oxysporum race 4 to produce a Musa acuminata plant with asilenced endogenous gene coding for susceptibility to Fusarium oxysporumrace 4. In some embodiments, the methods further comprise using the Musaacuminata plant with a silenced endogenous gene coding forsusceptibility to Fusarium oxysporum race 4 in a banana breedingprogram. In some embodiments, the methods of the present inventionutilize a plant cell that is the Musa acuminata plant cell with asilenced endogenous gene coding for susceptibility to Fusarium oxysporumrace 4. In some embodiments, the double-strand break is induced by anuclease selected from the group consisting of a TALEN, a meganuclease,a zinc finger nuclease, and a CRISPR-associated nuclease. In someembodiments, the double-strand break is induced by a CRISPR-associatednuclease and where a guide RNA is provided.

In some embodiments, the present invention provides methods forproducing a plant cell resistant to Fusarium oxysporum race 4 comprisingintroducing at least one genetic modification into one or moreendogenous nucleic acid sequences coding for susceptibility to Fusariumoxysporum race 4, wherein the genetic modification confers resistance toFusarium oxysporum race 4 to the plant cell. In some embodiments, atleast one genetic modification is introduced by a TALEN, a meganuclease,a zinc finger nuclease or a CRISPR-associated nuclease. In someembodiments, the at least one genetic modification is introduced by aCRISPR-associated nuclease and an associated guide RNA. In someembodiments, the at least one genetic modification is selected from thelist consisting of replacing a T corresponding to position 148 of SEQ IDNO: 14 with a G (148T>G), replacing a T corresponding to position 323 ofSEQ ID NO: 14 with an A (323T>A), replacing a G corresponding toposition 344 of SEQ ID NO: 14 with a C (344G>C), and replacing an Acorresponding to position 347 of SEQ ID NO: 14 with a T (347A>T). Insome embodiments, the at least one genetic modification results in achange in an amino acid selected from the group consisting of replacinga Leucine corresponding to position 50 of SEQ ID NO: 15 with a Valine(50L>V), replacing a Valine corresponding to position 108 of SEQ ID NO:15 with a Glutamic Acid (108V>E), replacing an Arginine corresponding toposition 115 of SEQ ID NO: 15 with a Proline (115R>P), and replacing anAspartic Acid corresponding to position 116 of SEQ ID NO: 15 with aValine (116D>V). In some embodiments, the plant cell is a Musa plantcell. In some embodiments, the plant cell is a Musa acuminata plantcell. In some embodiments, the methods further comprise producingtransformed plant tissue from the transformed plant cell. In someembodiments, the methods further comprise producing a transformedplantlet from the transformed plant tissue. In some embodiments, themethods further comprise producing a clone of the transformed plantlet.In some embodiments, the methods further comprise growing thetransformed plantlet or clone of the transformed plantlet into a maturetransformed plant. In some embodiments, the mature transformed plant isa Musa plant and the mature transformed Musa plant is capable ofproducing fruit. In some embodiments, the methods further compriseproducing clones of the mature transformed Musa plant. In someembodiments, the methods further comprise using the mature transformedMusa plant or clone of the mature transformed Musa plant in a breedingmethod.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates banana FusR1 coding sequences aligned. Initiation(start) and termination (stop) codons are underlined.

FusR1 nucleotide base substitutions between Musa species are bolded.Substitutions that code for replacement amino acid residues (i.e., arenonsynonymous) are shown in bolded font with an asterisk (*); silentsubstitutions are shown in bolded font with a dot (⋅). The first 96bases code for a leader peptide (shown in lower case) that is cleavedfrom the mature protein. This is known to be common for Bowman-Birkproteins (Barbosa et al., 2007). Inventor confirmed the extent of theleader sequence using two different bioinformatics tools, SignalP-5.0(Armenteros et al, 2019), and PrediSi (Hiller et al., 2004), which bothidentified the same leader peptide. Using the bioinformatics toolDeepLoc-1.0 (Armenteros et al., 2017), inventor then established thatthe mature FUSR1 protein is localized to the cell cytoplasm (likelihoodof 0.9732).

Bases shown in UPPER CASE code for the mature protein.

A missing base, shown as a dash (−), in the M. balbisiana FusR1 sequenceresults in a premature stop codon (shown in italicized, underlined lowercase), relative to the other FusR1 sequences. As described in the text,FusR1 mRNAs from all M. balbisiana accessions inventor examined have anunspliced (i.e., expressed) intron; for clarity in the Figure and tofocus on sequence similarities/differences in FusR1 coding sequencesfrom different banana species, the intron sequence has been removed herefrom M. balbisiana, even though inventor has not seen that happen. ThusSEQ ID NO: 27 is a ‘hypothetical” coding sequence.

The M. itinerans FusR1 sequence was obtained from multiple accessions(ITC1526, ITC1571, and PT-BA-00223), all of which are FW-resistant. TheM. acuminata FusR1 sequence labeled ‘FW-resistant’ was obtained frommultiple FW-resistant accessions, including ITC0896 (M. a. subspeciesbanksii) and PT-BA-00281 (Pisang Bangkahulu). The M. acuminata sequencelabeled ‘sensitive’ is from FW-sensitive accessions ITC0507, ITC0685,PT-BA-00304, PT-BA-00310, and PT-BA-00315. These accessions includemultiple samples from banana cultivars such as Pisang Madu, PisangPipit, and Pisang Rojo Uter, all of which have been well-characterizedas FW-sensitive (Chen et al, 2019). The M. balbisiana sequence includedhere was obtained from ITC1016. FusR1 from M. basjoo is fromFW-resistant accessions (ITC0061 and PD #3064).

Examination of FIG. 1 reveals that our FusR1 banana sequences arewell-conserved in the region that codes for the leader peptide, as isexpected. However, the FusR1 sequence that codes for the mature FUSR1protein shows an unusually high number of nonsynonymous substitutions.This is the result of severe selective pressure on these proteins, whichis reflected in the elevated Ka/Ks ratios seen for these genes. (Seebelow.) Inventor found 2 FW-resistant alleles for FusR1 from M.itinerans. These differ very slightly and for simplicity, only Allele 1(SEQ ID NO: 2) from M. itinerans is shown in FIG. 1. The Allele 2 codingsequence from M. itinerans is included in the Sequence Listing as SEQ IDNO: 5. Similarly, inventor found 2 FusR1 FW-resistant alleles in M.acuminata. These differ only by a single silent base substitution.Again, for simplicity, FIG. 1 shows only one of these alleles (SEQ IDNO: 9). The second allele, not shown in FIG. 1, is recorded in theSequence Listing as SEQ ID NO: 11.

FIG. 2 illustrates banana FUSR1 protein sequences aligned. Amino acidresidues that differ between the banana FUSR1 protein sequences areunderlined. The first 32 residues constitute a leader peptide which iscleaved from the mature protein. Leader sequence residues are shown inlower case, and mature protein residues in UPPER CASE.

The functional folded banana FUSR1 protein consists of two subdomains:Subdomain 1 is indicated by light grey shading; Subdomain 2 is indicatedby dark grey shading. As in other Bowman-Birk proteins, banana FUSR1structure is maintained by 14 disulfide bonds. The cysteine residuesthat form these disulfide bonds are shown in bold. Each subdomaincontains a reactive site, shown in italics. Residues that are specificfor trypsin (Subdomain 1) and chymotrypsin (Subdomain 2) are indicatedby an asterisk (*). For M. acuminata, residues that differ between theFoc4-sensitive FusR1 allele and the Foc-4 resistant alleles are shown bya dot (⋅), with the arginine residue (number 115) that explains Foc4sensitivity shown in bold font with a dot (⋅).

FIG. 3 provides a phylogenetic tree for several banana species, based onnucleotide sequences of the C2H2 gene.

The tree topology shown here was recovered from analysis of our bananaC2H2 nucleotide sequences. This topology is identical to that recoveredfrom analysis of the C2H2 protein sequence. The same tree was recoveredfrom our TOPO6 nucleotide and protein sequences. The topology shown hereis also similar to that in references.

It is important note that in contrast, topologies recovered from theFusR1 protein sequences and the protein-coding regions of the FusR1 genegive a different topology, which is clearly the result of the selectivepressures imposed on FusR1 during adaptation due to challenge byFusarium. The non-coding regions of FusR1 have the same topology as thephylogenetic trees for C2H2 and TOPO6.

The evolutionary history was inferred using the Maximum Parsimonymethod. The single most parsimonious tree is shown. The consistencyindex is 1.000000, the retention index is 1.000000, and the compositeindex is 1.000000 for all sites. The MP tree was obtained using theSubtree-Pruning-Regrafting (SPR) algorithm with search level 0 in whichthe initial trees were obtained by the random addition of sequences (10replicates). This analysis involved 5 nucleotide sequences. Codonpositions included were 1st+2nd+3rd+Noncoding. All positions with lessthan 95% site coverage were eliminated, i.e., fewer than 5% alignmentgaps, missing data, and ambiguous bases were allowed at any position(partial deletion option). There were a total of 218 positions in thefinal dataset. Evolutionary analyses were conducted in MEGA X (Kumar etal. 2018).

FIG. 4 provides a phylogenetic tree for several banana species, based onFUSR1 protein sequences. Note that this tree unites Musa acuminata andM. basjoo, in contrast to their actual phylogenetic relationship. M.acuminata is most closely related to M. balbisiana, with M. basjoo as asister taxon to these 2 species. However, because of the severe effectsof positive selection, the FusR1 protein sequence of M. acuminata and M.basjoo cluster together. (In fact, these protein sequences areidentical.)

The evolutionary history was inferred using the Maximum Parsimonymethod. The single most parsimonious tree with length=55 is shown. Theconsistency index is 0.963636, the retention index is 0.875000, and thecomposite index is 0.843182 for all sites. The MP tree was obtainedusing the Subtree-Pruning-Regrafting (SPR) algorithm with search level 0in which the initial trees were obtained by the random addition ofsequences (10 replicates). Evolutionary analyses were conducted in MEGAX.

FIG. 5 provides the alignment of FusR1 mRNA sequences from FW-sensitiveMusa balbisiana accessions. The sequences included here were obtainedfrom many M. balbisiana accessions, including ITC1016, ITC0545, ITC0080,ITC1527, ITC0565, ITC1781, ITC1580, and several others.

FusR1 nucleotide base substitutions between Musa balbisiana accessionsare in italics. Start and termination (stop) codons are shown in lowercase. Insertions, relative to other M. balbisiana accessions (as well asto FusR1 sequences from all other plants inventor analyzed), are bolded.Nucleotide deletions are shown by the colon symbol (:). The 85 base pairdeletion in FusR1 from accessions ITC0545 and ITC1781 is unique to M.balbisiana. As the sequence of FusR1 from ITC1781 is identical to thatfrom ITC0545, ITC1781 is not presented in FIG. 5. Similarly, the singlebase pair deletion found in these FW-sensitive M. balbisiana accessionshas not been found in any other FusR1 sequence. However it exists in allM. balbisiana accessions inventor analyzed. This single base pairdeletion results in a premature termination codon relative to the FusR1sequences from FW-resistant banana accessions.

All M. balbisiana accessions inventor examined had one of the 4 alleletypes shown here. Several accessions shared identical FusR1 alleles.Thus, for simplicity, only 4 accessions are shown in this figure. These4 FusR1 alleles are all very similar in nucleotide sequence. There aretranscriptional variants between accessions but all these variants havethe expressed, non-spliced intron. All accessions also have the singlebase pair base pair deletion. Three accessions also have an 85 base pairdeletion, and several have a 4 base pair insertion.

Thus all these FusR1 sequences are ‘broken’ and they all code fornon-functional FusR1 proteins. Significantly, all these M. balbisianaaccessions are FW-sensitive.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure provides a solution of fungal, viral, bacterialand/or nematode diseases by inducing a defense response to many invadingpathogens. The present disclosure provides methods of identifyinggenetic materials that can drive disease resistance and/or fungalresistance in plants including banana and in plants and plant parts.Also, the present disclosure provides methods of transferring geneticmaterials to susceptible banana cultivars in order to give rise totraits of disease and/or fungal resistance. Furthermore, the presentdisclosure teaches newly-identified genetic components and methods ofgenerating genetically modified plants, plant cells, tissues and seeds,having modified disease resistance.

I. Definitions

Unless stated otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. While the following termsare believed to be well understood by one of ordinary skill in the art,the following definitions are set forth to facilitate explanation of thepresently disclosed subject matter. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present disclosure, preferred methods andmaterials are described. The following terms are defined below. Thesedefinitions are for illustrative purposes and are not intended to limitthe common meaning in the art of the defined terms.

The term “a” or “an” refers to one or more of that entity, i.e., canrefer to a plural referent. As such, the terms “a” or “an”, “one ormore” and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

As used in this specification, the term “and/or” is used in thisdisclosure to mean either “and” or “or” unless indicated otherwise.

Throughout this specification, unless the context requires otherwise,the words “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids. In some embodiments, a fragmentof a polypeptide or polynucleotide comprises at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the entirelength of the reference polypeptide or polynucleotide. In someembodiments, a polypeptide or polynucleotide fragment may contain 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000 or more nucleotides or amino acids.

As used herein, the term “codon optimization” implies that the codonusage of a DNA or RNA is adapted to that of a cell or organism ofinterest to improve the transcription rate of said recombinant nucleicacid in the cell or organism of interest. The skilled person is wellaware of the fact that a target nucleic acid can be modified at oneposition due to the codon degeneracy, whereas this modification willstill lead to the same amino acid sequence at that position aftertranslation, which is achieved by codon optimization to take intoconsideration the species-specific codon usage of a target cell ororganism.

As used herein, the term “endogenous” or “endogenous gene,” refers tothe naturally occurring gene, in the location in which it is naturallyfound within the host cell genome. “Endogenous gene” is synonymous with“native gene” as used herein. An endogenous gene as described herein caninclude alleles of naturally occurring genes that have been mutatedaccording to any of the methods of the present disclosure, i.e. anendogenous gene could have been modified at some point by traditionalplant breeding methods and/or next generation plant breeding methods.

As used herein, the term “exogenous” refers to a substance coming fromsome source other than its native source. For example, the terms“exogenous protein,” or “exogenous gene” refer to a protein or gene froma non-native source, and that has been artificially supplied to abiological system. As used herein, the term “exogenous” is usedinterchangeably with the term “heterologous,” and refers to a substancecoming from some source other than its native source.

The terms “genetically engineered host cell,” “recombinant host cell,”and “recombinant strain” are used interchangeably herein and refer tohost cells that have been genetically engineered by the methods of thepresent disclosure. Thus, the terms include a host cell (e.g., bacteria,yeast cell, fungal cell, CHO, human cell, plant cell, protoplast derivedfrom plant, callus, etc.) that has been genetically altered, modified,or engineered, such that it exhibits an altered, modified, or differentgenotype and/or phenotype (e.g., when the genetic modification affectscoding nucleic acid sequences), as compared to the naturally-occurringhost cell from which it was derived. It is understood that the termsrefer not only to the particular recombinant host cell in question, butalso to the progeny or potential progeny of such a host cell.

As used herein, the term “heterologous” refers to a substance comingfrom some source or location other than its native source or location.In some embodiments, the term “heterologous nucleic acid” refers to anucleic acid sequence that is not naturally found in the particularorganism. For example, the term “heterologous promoter” may refer to apromoter that has been taken from one source organism and utilized inanother organism, in which the promoter is not naturally found. However,the term “heterologous promoter” may also refer to a promoter that isfrom within the same source organism, but has merely been moved to anovel location, in which said promoter is not normally located.

Heterologous gene sequences can be introduced into a target cell byusing an “expression vector,” which can be a eukaryotic expressionvector, for example a plant expression vector. Methods used to constructvectors are well known to a person skilled in the art and described invarious publications. In particular, techniques for constructingsuitable vectors, including a description of the functional componentssuch as promoters, enhancers, termination and polyadenylation signals,selection markers, origins of replication, and splicing signals, arereviewed in the prior art. Vectors may include but are not limited toplasmid vectors, phagemids, cosmids, artificial/mini-chromosomes (e.g.ACE), or viral vectors such as baculovirus, retrovirus, adenovirus,adeno-associated virus, herpes simplex virus, retroviruses,bacteriophages. The eukaryotic expression vectors will typically containalso prokaryotic sequences that facilitate the propagation of the vectorin bacteria such as an origin of replication and antibiotic resistancegenes for selection in bacteria. A variety of eukaryotic expressionvectors, containing a cloning site into which a polynucleotide can beoperatively linked, are well known in the art and some are commerciallyavailable from companies such as Stratagene, La Jolla, Calif.;Invitrogen, Carlsbad, Calif.; Promega, Madison, Wis. or BD BiosciencesClontech, Palo Alto, Calif. In one embodiment the expression vectorcomprises at least one nucleic acid sequence which is a regulatorysequence necessary for transcription and translation of nucleotidesequences that encode for a peptide/polypeptide/protein of interest.

As used herein, the term “naturally occurring” as applied to a nucleicacid, a polypeptide, a cell, or an organism, refers to a nucleic acid,polypeptide, cell, or organism that is found in nature. The term“naturally occurring” may refer to a gene or sequence derived from anaturally occurring source. Thus, for the purposes of this disclosure, a“non-naturally occurring” sequence is a sequence that has beensynthesized, mutated, engineered, edited, or otherwise modified to havea different sequence from known natural sequences. In some embodiments,the modification may be at the protein level (e.g., amino acidsubstitutions). In other embodiments, the modification may be at the DNAlevel (e.g., nucleotide substitutions).

As used herein, the term “nucleotide change” or “nucleotidemodification” refers to, e.g., nucleotide substitution, deletion, and/orinsertion, as is well understood in the art. For example, suchnucleotide changes/modifications include mutations containingalterations that produce silent substitutions, additions, or deletions,but do not alter the properties or activities of the encoded protein orhow the proteins are made. As another example, such nucleotidechanges/modifications include mutations containing alterations thatproduce replacement substitutions, additions, or deletions, that alterthe properties or activities of the encoded protein or how the proteinsare made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

The term “next generation plant breeding” refers to a host of plantbreeding tools and methodologies that are available to today's breeder.A key distinguishing feature of next generation plant breeding is thatthe breeder is no longer confined to relying upon observed phenotypicvariation, in order to infer underlying genetic causes for a giventrait. Rather, next generation plant breeding may include theutilization of molecular markers and marker assisted selection (MAS),such that the breeder can directly observe movement of alleles andgenetic elements of interest from one plant in the breeding populationto another, and is not confined to merely observing phenotype. Further,next generation plant breeding methods are not confined to utilizingnatural genetic variation found within a plant population. Rather, thebreeder utilizing next generation plant breeding methodology can accessa host of modern genetic engineering tools that directlyalter/change/edit the plant's underlying genetic architecture in atargeted manner, in order to bring about a phenotypic trait of interest.In aspects, the plants bred with a next generation plant breedingmethodology are indistinguishable from a plant that was bred in atraditional manner, as the resulting end product plant couldtheoretically be developed by either method. In particular aspects, anext generation plant breeding methodology may result in a plant thatcomprises: a genetic modification that is a deletion or insertion of anysize; a genetic modification that is one or more base pair substitution;a genetic modification that is an introduction of nucleic acid sequencesfrom within the plant's natural gene pool (e.g. any plant that could becrossed or bred with a plant of interest) or from editing of nucleicacid sequences in a plant to correspond to a sequence known to occur inthe plant's natural gene pool; and offspring of said plants.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of thedisclosure can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

The terms “polynucleotide,” “nucleic acid,” and “nucleotide sequence,”used interchangeably herein, refers to a polymeric form of nucleotidesof any length, either ribonucleotides or deoxyribonucleotides, oranalogs thereof. This term refers to the primary structure of themolecule, and thus includes double- and single-stranded DNA, as well asdouble- and single-stranded RNA. This term includes, but is not limitedto, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. It also includes modified nucleic acidssuch as methylated and/or capped nucleic acids, nucleic acids containingmodified bases, backbone modifications, and the like. “Oligonucleotide”generally refers to polynucleotides of between about 5 and about 100nucleotides of single- or double-stranded DNA. However, for the purposesof this disclosure, there is no upper limit to the length of anoligonucleotide. Oligonucleotides are also known as “oligomers” or“oligos” and may be isolated from genes, or chemically synthesized bymethods known in the art. The terms “polynucleotide” “nucleic acid,” and“nucleotide sequence” should be understood to include, as applicable tothe embodiments being described, single-stranded (such as sense orantisense) and double-stranded polynucleotides.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the disclosure. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. Vectors can be plasmids, viruses,bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. As used herein, the term“expression” refers to the production of a functional end-product e.g.,an mRNA or a protein (precursor or mature).

The term “traditional plant breeding” refers to the utilization ofnatural variation found within a plant population as a source foralleles and genetic variants that impart a trait of interest to a givenplant. Traditional breeding methods make use of crossing procedures thatrely largely upon observed phenotypic variation to infer causativeallele association. That is, traditional plant breeding relies uponobservations of expressed phenotype of a given plant to infer underlyinggenetic cause. These observations are utilized to inform the breedingprocedure in order to move allelic variation into germplasm of interest.Further, traditional plant breeding has also been characterized ascomprising random mutagenesis techniques, which can be used to introducegenetic variation into a given germplasm. These random mutagenesistechniques may include chemical and/or radiation-based mutagenesisprocedures. Consequently, one key feature of traditional plant breeding,is that the breeder does not utilize a genetic engineering tool thatdirectly alters/changes/edits the plant's underlying geneticarchitecture in a targeted manner, in order to introduce geneticdiversity and bring about a phenotypic trait of interest.

A “CRISPR-associated effector” as used herein can thus be defined as anynuclease, nickase, or recombinase associated with the CRISPR (ClusteredRegularly Interspaced Short Palindromic Repeats), having the capacity tointroduce a single- or double-strand cleavage into a genomic targetsite, or having the capacity to introduce a targeted modification,including a point mutation, an insertion, or a deletion, into a genomictarget site of interest. At least one CRISPR-associated effector can acton its own, or in combination with other molecules as part of amolecular complex. The CRISPR-associated effector can be present asfusion molecule, or as individual molecules associating by or beingassociated by at least one of a covalent or non-covalent interactionwith gRNA and/or target site so that the components of theCRISPR-associated complex are brought into close physical proximity.

A “base editor” as used herein refers to a protein or a fragment thereofhaving the same catalytic activity as the protein it is derived from,which protein or fragment thereof, alone or when provided as molecularcomplex, referred to as base editing complex herein, has the capacity tomediate a targeted base modification, i.e., the conversion of a base ofinterest resulting in a point mutation of interest, which in turn canresult in a targeted mutation, if the base conversion does not cause asilent mutation, but rather a conversion of an amino acid encoded by thecodon comprising the position to be converted with the base editor. Atleast one base editor according to the present disclosure temporarily orpermanently linked to at least one CRISPR-associated effector, oroptionally to a component of at least one CRISPR-associated effectorcomplex.

The term “Cas9 nuclease” and “Cas9” can be used interchangeably herein,which refer to a RNA-guided DNA endonuclease enzyme associated with theCRISPR (Clustered Regularly Interspaced Short Palindromic Repeats),including the Cas9 protein or fragments thereof (such as a proteincomprising an active DNA cleavage domain of Cas9 and/or a gRNA bindingdomain of Cas9). Cas9 is a component of the CRISPR/Cas genome editingsystem, which targets and cleaves a DNA target sequence to form a DNAdouble strand breaks (DSB) under the guidance of a guide RNA.

The term “CRISPR RNA” or “crRNA” refers to the RNA strand responsiblefor hybridizing with target DNA sequences, and recruiting CRISPRendonucleases and/or CRISPR-associated effectors. crRNAs may benaturally occurring, or may be synthesized according to any known methodof producing RNA.

The term “tracrRNA” refers to a small trans-encoded RNA. TracrRNA iscomplementary to and base pairs with crRNA to form a crRNA/tracrRNAhybrid, capable of recruiting CRISPR endonucleases and/orCRISPR-associated effectors to target sequences.

The term “Guide RNA” or “gRNA” as used herein refers to an RNA sequenceor combination of sequences capable of recruiting a CRISPR endonucleaseand/or CRISPR-associated effectors to a target sequence. Typically gRNAis composed of crRNA and tracrRNA molecules forming complexes throughpartial complement, wherein crRNA comprises a sequence that issufficiently complementary to a target sequence for hybridization anddirects the CRISPR complex (i.e. Cas9-crRNA/tracrRNA hybrid) tospecifically bind to the target sequence. Also, single guide RNA (sgRNA)can be designed, which comprises the characteristics of both crRNA andtracrRNA. Therefore, as used herein, a guide RNA can be a natural orsynthetic crRNA (e.g., for Cpf1), a natural or synthetic crRNA/tracrRNAhybrid (e.g., for Cas9), or a single-guide RNA (sgRNA).

The term “guide sequence” or “spacer sequence” refers to the portion ofa crRNA or guide RNA (gRNA) that is responsible for hybridizing with thetarget DNA.

The term “protospacer” refers to the DNA sequence targeted by a guidesequence of crRNA or gRNA. In some embodiments, the protospacer sequencehybridizes with the crRNA or gRNA guide (spacer) sequence of a CRISPRcomplex.

The term “CRISPR landing site” as used herein, refers to a DNA sequencecapable of being targeted by a CRISPR-Cas complex. In some embodiments,a CRISPR landing site comprises a proximately placedprotospacer/Protopacer Adjacent Motif combination sequence that iscapable of being cleaved by a CRISPR complex.

The term “CRISPR complex”, “CRISPR endonuclease complex”, “CRISPR Cascomplex”, or “CRISPR-gRNA complex” are used interchangeably herein.“CRISPR complex” refers to a Cas9 nuclease and/or a CRISPR-associatedeffectors complexed with a guide RNA (gRNA). The term “CRISPR complex”thus refers to a combination of CRISPR endonuclease and guide RNAcapable of inducing a double stranded break at a CRISPR landing site. Insome embodiments, “CRISPR complex” of the present disclosure refers to acombination of catalytically dead Cas9 protein and guide RNA capable oftargeting a target sequence, but not capable of inducing a doublestranded break at a CRISPR landing site because it loses a nucleaseactivity. In other embodiments, “CRISPR complex” of the presentdisclosure refers to a combination of Cas9 nickase and guide RNA capableof introducing gRNA-targeted single-strand breaks in DNA instead of thedouble-strand breaks created by wild type Cas enzymes.

As used herein, the term “directing sequence-specific binding” in thecontext of CRISPR complexes refers to a guide RNA's ability to recruit aCRISPR endonuclease and/or a CRISPR-associated effectors to a CRISPRlanding site.

As used herein, the term “deaminase” refers to an enzyme that catalyzesthe deamination reaction. In some embodiments of the present disclosure,the deaminase refers to a cytidine deaminase, which catalyzes thedeamination of a cytidine or a deoxycytidine to a uracil or adeoxyuridine, respectively. In other embodiments of the presentdisclosure, the deaminase refers to an adenosine deaminase, whichcatalyzes the deamination of an adenine to form hypoxanthine (in theform of its nucleoside inosine), which is read as guanine by DNApolymerase.

As used herein, the term “glycosylase” refers to a family of enzymesinvolved in base excision repair, classified under EC number EC 3.2.2.Base excision repair is the mechanism by which damaged bases in DNA areremoved and replaced. DNA glycosylases catalyze the first step of thisprocess. They remove the damaged nitrogenous base while leaving thesugar-phosphate backbone intact, creating an apurinic/apyrimidinic site,commonly referred to as an AP site. This is accomplished by flipping thedamaged base out of the double helix followed by cleavage of theN-glycosidic bond. In some embodiments of the present disclosure, in anexpectation of affording a mutation introduction tendency different fromthat of deaminase and the like, a base excision reaction by hydrolysisof N-glycosidic bond of DNA, and then inducing mutation introduction ina repair process of cells is used. In aspects, an enzyme havingcytosine-DNA glycosylase (CDG) activity or thymine-DNA glycosylase (TDG)activity is used. In aspects, a mutant of yeast mitochondrial uracil-DNAglycosylase (UNG 1), is used as an enzyme that performs such baseexcision reaction. Nishida et al., US 2017/0321210 A1, published on Nov.9, 2017, is incorporated by reference herein.

As used herein the term “targeted” refers to the expectation that oneitem or molecule will interact with another item or molecule with adegree of specificity, so as to exclude non-targeted items or molecules.For example, a first polynucleotide that is targeted to a secondpolynucleotide, according to the present disclosure has been designed tohybridize with the second polynucleotide in a sequence specific manner(e.g., via Watson-Crick base pairing). In some embodiments, the selectedregion of hybridization is designed so as to render the hybridizationunique to the one, or more targeted regions. A second polynucleotide cancease to be a target of a first targeting polynucleotide, if itstargeting sequence (region of hybridization) is mutated, or is otherwiseremoved/separated from the second polynucleotide. Furthermore,“targeted” can be interchangeably used with “site-specific” or“site-directed,” which refers to an action of molecular biology whichuses information on the sequence of a genomic region of interest to bemodified, and which further relies on information of the mechanism ofaction of molecular tools, e.g., nucleases, including CRISPR nucleasesand variants thereof, TALENs, ZFNs, meganucleases or recombinases,DNA-modifying enzymes, including base modifying enzymes like cytidinedeaminase enzymes, histone modifying enzymes and the like, DNA-bindingproteins, cr/tracr RNAs, guide RNAs and the like.

The term “seed region” refers to the critical portion of a crRNA's orguide RNA's guide sequence that is most susceptible to mismatches withtheir targets. In some embodiments, a single mismatch in the seed regionof a crRNA/gRNA can render a CRISPR complex inactive at that bindingsite. In some embodiments, the seed regions for Cas9 endonucleases arelocated along the last ˜12 nts of the 3′ portion of the guide sequence,which correspond (hybridize) to the portion of the protospacer targetsequence that is adjacent to the PAM. In some embodiments, the seedregions for Cpf1 endonucleases are located along the first ˜5 nts of the5′ portion of the guide sequence, which correspond (hybridize) to theportion of the protospacer target sequence adjacent to the PAM.

The term “sequence identity” refers to the percentage of bases or aminoacids between two polynucleotide or polypeptide sequences that are thesame, and in the same relative position. As such one polynucleotide orpolypeptide sequence has a certain percentage of sequence identitycompared to another polynucleotide or polypeptide sequence. For sequencecomparison, typically one sequence acts as a reference sequence, towhich test sequences are compared. The term “reference sequence” refersto a molecule to which a test sequence is compared. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, Computer Applic. Biol. Sci., 4:11-17 (1988).

“Complementary” refers to the capacity for pairing, through basestacking and specific hydrogen bonding, between two sequences comprisingnaturally or non-naturally occurring bases or analogs thereof. Forexample, if a base at one position of a nucleic acid is capable ofhydrogen bonding with a base at the corresponding position of a target,then the bases are considered to be complementary to each other at thatposition. Nucleic acids can comprise universal bases, or inert abasicspacers that provide no positive or negative contribution to hydrogenbonding. Base pairings may include both canonical Watson-Crick basepairing and non-Watson-Crick base pairing (e.g., Wobble base pairing andHoogsteen base pairing). It is understood that for complementary basepairings, adenosine-type bases (A) are complementary to thymidine-typebases (T) or uracil-type bases (U), that cytosine-type bases (C) arecomplementary to guanosine-type bases (G), and that universal bases suchas such as 3-nitropyrrole or 5-nitroindole can hybridize to and areconsidered complementary to any A, C, U, or T. Nichols et al., Nature,1994; 369:492-493 and Loakes et al., Nucleic Acids Res., 1994;22:4039-4043. Inosine (I) has also been considered in the art to be auniversal base and is considered complementary to any A, C, U, or T. SeeWatkins and Santa Lucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267.

As referred to herein, a “complementary nucleic acid sequence” is anucleic acid sequence comprising a sequence of nucleotides that enablesit to non-covalently bind to another nucleic acid in asequence-specific, antiparallel, manner (i.e., a nucleic acidspecifically binds to a complementary nucleic acid) under theappropriate in vitro and/or in vivo conditions of temperature andsolution ionic strength.

Methods of sequence alignment for comparison and determination ofpercent sequence identity and percent complementarity are well known inthe art. Optimal alignment of sequences for comparison can be conducted,e.g., by the homology alignment algorithm of Needleman and Wunsch,(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), by manual alignment and visualinspection (see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology), by use of algorithms know in the art including theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,(1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol.Biol. 215:403-410, respectively. Software for performing BLAST analysesis publicly available through the National Center for BiotechnologyInformation. Some alignment programs are MacVector (Oxford MolecularLtd, Oxford, U.K.), ALIGN Plus (Scientific and Educational Software,Pennsylvania) and AlignX (Vector NTI, Invitrogen, Carlsbad, Calif.).Another alignment program is Sequencher (Gene Codes, Ann Arbor, Mich.),using default parameters, and MUSCLE (Multiple Sequence Comparison byLog-Expection; a computer software licensed as public domain).

Herein, the term “hybridize” refers to pairing between complementarynucleotide bases (e.g., adenine (A) forms a base pair with thymine (T)in a DNA molecule and with uracil (U) in an RNA molecule, and guanine(G) forms a base pair with cytosine (C) in both DNA and RNA molecules)to form a double-stranded nucleic acid molecule. (See, e.g., Wahl andBerger (1987) Methods Enzymol. 152:399; Kimmel, (1987) Methods Enzymol.152:507). In addition, it is also known in the art that forhybridization between two RNA molecules (e.g., dsRNA), guanine (G) basepairs with uracil (U). For example, G/U base-pairing is partiallyresponsible for the degeneracy (i.e., redundancy) of the genetic code inthe context of tRNA anti-codon base-pairing with codons in mRNA. In thecontext of this disclosure, a guanine (G) of a protein-binding segment(dsRNA duplex) of a guide RNA molecule is considered complementary to auracil (U), and vice versa. As such, when a G/U base-pair can be made ata given nucleotide position a protein-binding segment (dsRNA duplex) ofa guide RNA molecule, the position is not considered to benon-complementary, but is instead considered to be complementary. It isunderstood in the art that the sequence of polynucleotide need not be100% complementary to that of its target nucleic acid to be specificallyhybridizable. Moreover, a polynucleotide may hybridize over one or moresegments such that intervening or adjacent segments are not involved inthe hybridization event (e.g., a loop structure or hairpin structure). Apolynucleotide can comprise at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or 100% sequence complementarity to a targetregion within the target nucleic acid sequence to which they aretargeted.

The term “modified” refers to a substance or compound (e.g., a cell, apolynucleotide sequence, and/or a polypeptide sequence) that has beenaltered or changed as compared to the corresponding unmodified substanceor compound.

“Isolated” refers to a material that is free to varying degrees fromcomponents which normally accompany it as found in its native state.

The term “gene edited plant, part or cell” as used herein refers to aplant, part or cell that comprises one or more endogenous genes that areedited by a gene editing system. The gene editing system of the presentdisclosure comprises a targeting element and/or an editing element. Thetargeting element is capable of recognizing a target genomic sequence.The editing element is capable of modifying the target genomic sequence,e.g., by substitution or insertion of one or more nucleotides in thegenomic sequence, deletion of one or more nucleotides in the genomicsequence, alteration of genomic sequences to include regulatorysequences, insertion of transgenes at a safe harbor genomic site orother specific location in the genome, or any combination thereof. Thetargeting element and the editing element can be on the same nucleicacid molecule or different nucleic acid molecules. In some embodiments,the editing element is capable of precise genome editing by substitutionof a single nucleotide using a base editor, such cytosine base editor(CBE) and/or adenine base editor (ABE), which is directly or indirectlyfused to a CRISPR-associated effector protein.

The term “plant” refers to whole plants. The term “plant part” includedifferentiated and undifferentiated tissues including, but not limitedto: plant organs, plant tissues, roots, stems, shoots, rootstocks,scions, stipules, petals, leaves, flowers, ovules, pollens, bracts,petioles, internodes, bark, pubescence, tillers, rhizomes, fronds,blades, stamens, fruits, seeds, tumor tissue and plant cells (e.g.,single cells, protoplasts, embryos, and callus tissue). Plant cellsinclude, without limitation, cells from seeds, suspension cultures,embryos, meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores. The plant tissue maybe in a plant or in a plant organ, tissue or cell culture.

As used herein when discussing plants, the term “ovule” refers to thefemale gametophyte, whereas the term “pollen” means the malegametophyte.

As used herein, the term “plant tissue” refers to any part of a plant.Examples of plant organs include, but are not limited to the leaf, stem,root, tuber, seed, branch, pubescence, nodule, leaf axil, flower,pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract,fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone,rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen,and leaf sheath.

As used herein, the term “phenotype” refers to the observable charactersof an individual cell, cell culture, organism (e.g., a plant), or groupof organisms which results from the interaction between thatindividual's genetic makeup (i.e., genotype) and the environment.

The terms “transgene” or “transgenic” as used herein refer to at leastone nucleic acid sequence that is taken from the genome of one organism,or produced synthetically, and which is then introduced into a host cellor organism or tissue of interest and which is subsequently integratedinto the host's genome by means of “stable” transformation ortransfection approaches. In contrast, the term “transient”transformation or transfection or introduction refers to a way ofintroducing molecular tools including at least one nucleic acid (DNA,RNA, single-stranded or double-stranded or a mixture thereof) and/or atleast one amino acid sequence, optionally comprising suitable chemicalor biological agents, to achieve a transfer into at least onecompartment of interest of a cell, including, but not restricted to, thecytoplasm, an organelle, including the nucleus, a mitochondrion, avacuole, a chloroplast, or into a membrane, resulting in transcriptionand/or translation and/or association and/or activity of the at leastone molecule introduced without achieving a stable integration orincorporation and thus inheritance of the respective at least onemolecule introduced into the genome of a cell. The terms“transgene-free” refers to a condition that transgene is not present orfound in the genome of a host cell or tissue or organism of interest.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary buds,ovaries, seed coat, endosperm, hypocotyls, cotyledons and the like. Theterm “plant organ” refers to plant tissue or a group of tissues thatconstitute a morphologically and functionally distinct part of a plant.“Progeny” comprises any subsequent generation of a plant.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference.

As used herein, the term “AGAMOUS Clade Transcription Factor” or “AGclade transcription factor” is a member of the AGAMOUS (AG) subfamily ofMIKC-type MADS-box genes. “MIKC-type” proteins represent a class ofMADS-domain transcription factors and are defined by a unique domainstructure: (1) ‘M’—a highly conserved DNA-binding MADS-domain, (2)‘I’—an intervening domain, (3) ‘K’—a keratin-like K-domain, and (4)‘C’—a C-terminal domain. In some embodiments, “AGAMOUS CladeTranscription Factor” or “AG clade transcription factor” furthercomprises an N-terminal region. In further embodiments, “AGAMOUS CladeTranscription Factor” or “AG clade transcription factor” comprises AG,SHP1, SHP2, and STK genes in plants of the present disclosure, each ofwhich has a NN motif in the M domain, a YQQ motif in the K domain,and/or a R/Q (R or Q) in the C domain.

By “biologically active portion” is meant a portion of a full-lengthparent peptide or polypeptide which portion retains an activity of theparent molecule. For example, a biologically active portion ofpolypeptide of the disclosure will retain the ability to confer diseaseresistance, especially resistance to fungal pathogens such as Fusarium.As used herein, the term “biologically active portion” includes deletionmutants and peptides, for example of at least about 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70,80, 90, 100, 120, 150, 300, 400, 500, 600, 700, 800, 900 or 1000contiguous amino acids, which comprise an activity of a parent molecule.Portions of this type may be obtained through the application ofstandard recombinant nucleic acid techniques or synthesized usingconventional liquid or solid phase synthesis techniques. For example,reference may be made to solution synthesis or solid phase synthesis asdescribed, for example, in Chapter 9 entitled “Peptide Synthesis” byAtherton and Shephard which is included in a publication entitled“Synthetic Vaccines” edited by Nicholson and published by BlackwellScientific Publications. Alternatively, peptides can be produced bydigestion of a peptide or polypeptide of the disclosure with proteinasessuch as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease.The digested fragments can be purified by, for example, high performanceliquid chromatographic (HPLC) techniques. Recombinant nucleic acidtechniques can also be used to produce such portions.

By “corresponds to” or “corresponding to” is meant a polynucleotide (a)having a nucleotide sequence that is substantially identical orcomplementary to all or a portion of a reference polynucleotide sequenceor (b) encoding an amino acid sequence identical to an amino acidsequence in a peptide or protein. This phrase also includes within itsscope a peptide or polypeptide having an amino acid sequence that issubstantially identical to a sequence of amino acids in a referencepeptide or protein.

The terms “growing” or “regeneration” as used herein mean growing awhole, differentiated plant from a plant cell, a group of plant cells, aplant part (including seeds), or a plant piece (e.g., from a protoplast,callus, or tissue part).

As used herein, the term “derived from” refers to the origin or source,and may include naturally occurring, recombinant, unpurified, orpurified molecules. A nucleic acid or an amino acid derived from anorigin or source may have all kinds of nucleotide changes or proteinmodification as defined elsewhere herein.

By “obtained from” is meant that a sample such as, for example, anucleic acid extract or polypeptide extract is isolated from, or derivedfrom, a particular source. For example, the extract may be isolateddirectly from plants, especially monocotyledonous plants and moreespecially non-graminaceous monocotyledonous plants such as banana.

The term “pathogen” is used herein in its broadest sense to refer to anorganism or an infectious agent whose infection of cells of viable planttissue elicits a disease response.

By “variant” polypeptide is intended a polypeptide derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins encompassedby the present disclosure are biologically active, that is they continueto possess the desired biological activity of the native protein, thatis, modulating or regulatory activity as described herein. Such variantsmay result from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a native R protein of thedisclosure will have at least 40%, 50%, 60%, 70%, generally at least75%, 80%, 85%, preferably about 90% to 95% or more, and more preferablyabout 98% or more sequence identity to the amino acid sequence for thenative protein as determined by sequence alignment programs describedelsewhere herein using default parameters. A biologically active variantof a protein of the disclosure may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2, or even 1 amino acid residue.

The proteins of the disclosure may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the R proteins can be preparedby mutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferable.

Individual substitutions deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother, Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine I,Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),Asparagine (N), Glutamine (Q). See also, Creighton, 1984. In addition,individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids in anencoded sequence are also “conservatively modified variations.”

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA, in the senseor antisense direction. The expression cassette comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression cassette may also be one which isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. The expression of the nucleotide sequencein the expression cassette may be under the control of a constitutivepromoter or of an inducible promoter which initiates transcription onlywhen the host cell is exposed to some particular external stimulus. Inthe case of a multicellular organism, the promoter can also be specificto a particular tissue or organ or stage of development in animal and/orplant including banana species.

As used herein, the term “vector”, “plasmid”, or “construct” refersbroadly to any plasmid or virus encoding an exogenous nucleic acid. Theterm should also be construed to include non-plasmid and non-viralcompounds which facilitate transfer of nucleic acid into virions orcells, such as, for example, polylysine compounds and the like. Thevector may be a viral vector that is suitable as a delivery vehicle fordelivery of the nucleic acid, or mutant thereof, to a cell, or thevector may be a non-viral vector which is suitable for the same purpose.Examples of viral and non-viral vectors for delivery of DNA to cells andtissues are well known in the art and are described, for example, in Maet al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples ofviral vectors include, but are not limited to, recombinant plantviruses. Non-limiting examples of plant viruses include, TMV-mediated(transient) transfection into tobacco (Tuipe, T-H et al (1993), J.Virology Meth, 42: 227-239), ssDNA genomes viruses (e.g., familyGeminiviridae), reverse transcribing viruses (e.g., familiesCaulimoviridae, Pseudoviridae, and Metaviridae), dsNRA viruses (e.g.,families Reoviridae and Partitiviridae), (−) ssRNA viruses (e.g.,families Rhabdoviridae and Bunyaviridae), (+) ssRNA viruses (e.g.,families Bromoviridae, Closteroviridae, Comoviridae, Luteoviridae,Potyviridae, Sequiviridae and Tombusviridae) and viroids (e.g., familiesPospiviroldae and Avsunviroidae). Detailed classification information ofplant viruses can be found in Fauquet et al (2008, “Geminivirus straindemarcation and nomenclature”. Archives of Virology 153:783-821,incorporated herein by reference in its entirety), and Khan et al.(Plant viruses as molecular pathogens; Publisher Routledge, 2002, ISBN1560228954, 9781560228950). Examples of non-viral vectors include, butare not limited to, liposomes, polyamine derivatives of DNA, and thelike.

Also, “vector” is defined to include, inter alia, any plasmid, cosmid,phage or Agrobacterium binary vector in double or single stranded linearor circular form which may or may not be self-transmissible ormobilizablez, and which can transform prokaryotic or eukaryotic hosteither by integration into the cellular genome or existextrachromosomally (e.g. autonomous replicating plasmid with an originof replication).

Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from actinomycetes and relatedspecies, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast orfungal cells).

Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, e.g. bacterial, orplant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of genomic DNA, this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance, hygromycin resistance or ampicillin resistance.

As used herein, the term “resistant”, or “resistance”, describes aplant, line or cultivar that shows fewer or reduced symptoms to a bioticpest or pathogen than a susceptible (or more susceptible) plant, line orvariety to that biotic pest or pathogen. These terms are variouslyapplied to describe plants that show no symptoms as well as plantsshowing some symptoms but that are still able to produce marketableproduct with an acceptable yield. Some lines that are referred to asresistant are only so in the sense that they may still produce a crop,even though the plants may appear visually stunted and the yield isreduced compared to uninfected plants. As defined by the InternationalSeed Federation (ISF), a non-governmental, non-profit organizationrepresenting the seed industry (see “Definition of the Terms Describingthe Reaction of Plants to Pests or Pathogens and to Abiotic Stresses forthe Vegetable Seed Industry”, May 2005), the recognition of whether aplant is affected by or subject to a pest or pathogen can depend on theanalytical method employed. Resistance is defined by the ISF as theability of plant types to restrict the growth and development of aspecified pest or pathogen and/or the damage they cause when compared tosusceptible plant varieties under similar environmental conditions andpest or pathogen pressure. Resistant plant types may still exhibit somedisease symptoms or damage. Two levels of resistance are defined. Theterm “high/standard resistance” is used for plant varieties that highlyrestrict the growth and development of the specified pest or pathogenunder normal pest or pathogen pressure when compared to susceptiblevarieties. “Moderate/intermediate resistance” is applied to plant typesthat restrict the growth and development of the specified pest orpathogen, but exhibit a greater range of symptoms or damage compared toplant types with high resistance. Plant types with intermediateresistance will show less severe symptoms than susceptible plantvarieties, when grown under similar field conditions and pathogenpressure. Methods of evaluating resistance are well known to one skilledin the art. Such evaluation may be performed by visual observation of aplant or a plant part (e.g., leaves, roots, flowers, fruits et. al) indetermining the severity of symptoms. For example, when each plant isgiven a resistance score on a scale of 1 to 5 based on the severity ofthe reaction or symptoms, with 1 being the resistance score applied tothe most resistant plants (e.g., no symptoms, or with the leastsymptoms), and 5 the score applied to the plants with the most severesymptoms, then a line is rated as being resistant when at least 75% ofthe plants have a resistance score at a 1, 2, or 3 level, whilesusceptible lines are those having more than 25% of the plants scoringat a 4 or 5 level. If a more detailed visual evaluation is possible,then one can use a scale from 1 to 10 so as to broaden out the range ofscores and thereby hopefully provide a greater scoring spread among theplants being evaluated.

Another scoring system is a root inoculation test based on thedevelopment of the necrosis after inoculation and its position towardsthe cotyledon (such as one derived from Bosland et al., 1991), wherein 0stands for no symptom after infection; 1 stands for a small necrosis atthe hypocotyl after infection; 2 stands a necrosis under the cotyledonsafter infection; 3 stands for necrosis above the cotyledons afterinfection; 4 stands for a necrosis above the cotyledons together with awilt of the plant after infection, while eventually, 5 stands for a deadplant.

In addition to such visual evaluations, disease evaluations can beperformed by determining the pathogen bio-density in a plant or plantpart using electron microscopy and/or through molecular biologicalmethods, such as protein hybridization (e.g., ELISA, measuring pathogenprotein density) and/or nucleic acid hybridization (e.g., RT-PCR,measuring pathogen RNA density). Depending on the particularpathogen/plant combination, a plant may be determined resistant to thepathogen, for example, if it has a pathogen RNA/DNA and/or proteindensity that is about 50%, or about 40%, or about 30%, or about 20%, orabout 10%, or about 5%, or about 2%, or about 1%, or about 0.1%, orabout 0.01%, or about 0.001%, or about 0.0001% of the RNA/DNA and/orprotein density in a susceptible plant.

Methods used in breeding plants for disease resistance are similar tothose used in breeding for other characters. It is necessary to know asmuch as possible about the nature of inheritance of the resistantcharacters in the host plant and the existence of physiological races orstrains of the pathogen.

As used herein, the term “full resistance” is referred to as completefailure of the pathogen to develop after infection, and may either bethe result of failure of the pathogen to enter the cell (no initialinfection) or may be the result of failure of the pathogen to multiplyin the cell and infect subsequent cells (no subliminal infection, nospread). The presence of full resistance may be determined byestablishing the absence of pathogen protein or pathogen RNA in cells ofthe plant, as well as the absence of any disease symptoms in said plant,upon exposure of said plant to an infective dosage of pathogen (i.e.after ‘infection’). Among breeders, this phenotype is often referred toas “immune”. “Immunity” as used herein thus refers to a form ofresistance characterized by absence of pathogen replication even whenthe pathogen is actively transferred into cells by e.g. electroporation.

As used herein, the term “partial resistance” is referred to as reducedmultiplication of the pathogen in the cell, as reduced (systemic)movement of the pathogen, and/or as reduced symptom development afterinfection. The presence of partial resistance may be determined byestablishing the systemic presence of low concentration of pathogenprotein or pathogen RNA in the plant and the presence of decreased ordelayed disease-symptoms in said plant upon exposure of said plant to aninfective dosage of pathogen. Protein concentration may be determined byusing a quantitative detection method (e.g. an ELISA method or aquantitative reverse transcriptase-polymerase chain reaction (RT-PCR)).Among breeders, this phenotype is often referred to as “intermediateresistant.”

As used herein, the term “tolerant” is used herein to indicate aphenotype of a plant wherein disease-symptoms remain absent uponexposure of said plant to an infective dosage of pathogen, whereby thepresence of a systemic or local pathogen infection, pathogenmultiplication, at least the presence of pathogen genomic sequences incells of said plant and/or genomic integration thereof can beestablished. Tolerant plants are therefore resistant for symptomexpression but symptomless carriers of the pathogen. Sometimes, pathogensequences may be present or even multiply in plants without causingdisease symptoms. This phenomenon is also known as “latent infection”.In latent infections, the pathogen may exist in a truly latentnon-infectious occult form, possibly as an integrated genome or anepisomal agent (so that pathogen protein cannot be found in thecytoplasm, while PCR protocols may indicate the present of pathogennucleic acid sequences) or as an infectious and continuously replicatingagent. A reactivated pathogen may spread and initiate an epidemic amongsusceptible contacts. The presence of a “latent infection” isindistinguishable from the presence of a “tolerant” phenotype in aplant.

As used herein, the term “susceptible” is used herein to refer to aplant having no or virtually no resistance to the pathogen resulting inentry of the pathogen into the plant and multiplication and systemicspread of the pathogen, resulting in disease symptoms. The term“susceptible” is therefore equivalent to “non-resistant”.

As used herein, the term “offspring” refers to any plant resulting asprogeny from a vegetative or sexual reproduction from one or more parentplants or descendants thereof. For instance an offspring plant may beobtained by cloning or selfing of a parent plant or by crossing twoparents plants and include selfings as well as the F1 or F2 or stillfurther generations. An F1 is a first-generation offspring produced fromparents at least one of which is used for the first time as donor of atrait, while offspring of second generation (F2) or subsequentgenerations (F3, F4, etc.) are specimens produced from selfings of F1's,F2's etc. An F1 may thus be (and usually is) a hybrid resulting from across between two true breeding parents (true-breeding is homozygous fora trait), while an F2 may be (and usually is) an offspring resultingfrom self-pollination of said F1 hybrids.

As used herein, the terms “dicotyledon,” “dicot” and “dicotyledonous”refer to a flowering plant having an embryo containing two seed halvesor cotyledons. Examples include tobacco; tomato; the legumes, includingpeas, alfalfa, clover and soybeans; oaks; maples; roses; mints;squashes; daisies; walnuts; cacti; violets and buttercups.

As used herein, the term “monocotyledon,” “monocot” or“monocotyledonous” refer to any of a subclass (Monocotyledoneae) offlowering plants having an embryo containing only one seed leaf andusually having parallel-veined leaves, flower parts in multiples ofthree, and no secondary growth in stems and roots. Examples includebanana, daffodils, sugarcane, ginger, lily, orchid, rice, corn, grasses,such as tall fescue, goat grass, and Kentucky bluegrass; grains, such aswheat, oats and barley, irises; onion and palm.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include nonexpressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “genotype” refers to the genetic makeup of anindividual cell, cell culture, tissue, organism (e.g., a plant), orgroup of organisms.

As used herein, the term “allele(s)” means any of one or morealternative forms of a gene, all of which alleles relate to at least onetrait or characteristic. In a diploid cell, the two alleles of a givengene occupy corresponding loci on a pair of homologous chromosomes.Since the present disclosure relates to QTLs, i.e. genomic regions thatmay comprise one or more genes or regulatory sequences, it is in someinstances more accurate to refer to “haplotype” (i.e. an allele of achromosomal segment) instead of “allele”, however, in those instances,the term “allele” should be understood to comprise the term “haplotype”.Alleles are considered identical when they express a similar phenotype.Differences in sequence are possible but not important as long as theydo not influence phenotype.

As used herein, the term “locus” (plural: “loci”) refers to any sitethat has been defined genetically. A locus may be a gene, or part of agene, or a DNA sequence that has some regulatory role, and may beoccupied by different sequences.

As used herein, the term “molecular marker” or “genetic marker” refersto an indicator that is used in methods for visualizing differences incharacteristics of nucleic acid sequences. Examples of such indicatorsare restriction fragment length polymorphism (RFLP) markers, amplifiedfragment length polymorphism (AFLP) markers, single nucleotidepolymorphisms (SNPs), insertion mutations, microsatellite markers(SSRs), sequence-characterized amplified regions (SCARs), cleavedamplified polymorphic sequence (CAPS) markers or isozyme markers orcombinations of the markers described herein which defines a specificgenetic and chromosomal location. Mapping of molecular markers in thevicinity of an allele is a procedure which can be performed quite easilyby the average person skilled in molecular-biological techniques whichtechniques are for instance described in Lefebvre and Chevre, 1995;Lorez and Wenzel, 2007, Srivastava and Narula, 2004, Meksem and Kahl,2005, Phillips and Vasil, 2001. General information concerning AFLPtechnology can be found in Vos et al. (1995, AFLP: a new technique forDNA fingerprinting, Nucleic Acids Res. 1995 November 11; 23(21):4407-4414).

As used herein, the term “hemizygous” refers to a cell, tissue ororganism in which a gene is present only once in a genotype, as a genein a haploid cell or organism, a sex-linked gene in the heterogameticsex, or a gene in a segment of chromosome in a diploid cell or organismwhere its partner segment has been deleted.

As used herein, the term “heterozygote” refers to a diploid or polyploidindividual cell or plant having different alleles (forms of a givengene) present at least at one locus.

As used herein, the term “heterozygous” refers to the presence ofdifferent alleles (forms of a given gene) at a particular gene locus.

As used herein, the term “homozygote” refers to an individual cell orplant having the same alleles at one or more loci.

As used herein, the term “homozygous” refers to the presence ofidentical alleles at one or more loci in homologous chromosomalsegments.

As used herein, the term “homologous” or “homolog” is known in the artand refers to related sequences that share a common ancestor or familymember and are determined based on the degree of sequence identity. Theterms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Homologsusually control, mediate, or influence the same or similar biochemicalpathways, yet particular homologs may give rise to differing phenotypes.It is therefore understood, as those skilled in the art will appreciate,that the disclosure encompasses more than the specific exemplarysequences. These terms describe the relationship between a gene found inone species, subspecies, variety, cultivar or strain and thecorresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this disclosure homologoussequences are compared.

The term “homolog” is sometimes used to apply to the relationshipbetween genes separated by the event of speciation (see “ortholog”) orto the relationship between genes separated by the event of geneticduplication (see “paralog”).

The term “homeolog” refers to a homeologous gene or chromosome,resulting from polyploidy or chromosomal duplication events. Thiscontrasts with the more common ‘homolog’, which is defined immediatelyabove.

The term “ortholog” refers to genes in different species that evolvedfrom a common ancestral gene by speciation. Normally, orthologs retainthe same function in the course of evolution. Identification oforthologs is critical for reliable prediction of gene function in newlysequenced genomes.

The term “paralog” refers to genes related by duplication within agenome. While orthologs generally retain the same function in the courseof evolution, paralogs can evolve new functions, even if these arerelated to the original one.

“Homologous sequences” or “homologs” or “orthologs” are thought,believed, or known to be functionally related. A functional relationshipmay be indicated in any one of a number of ways, including, but notlimited to: (a) degree of sequence identity and/or (b) the same orsimilar biological function. Preferably, both (a) and (b) are indicated.The degree of sequence identity may vary, but in one embodiment, is atleast 50% (when using standard sequence alignment programs known in theart), at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least 98.5%, orat least about 99%, or at least 99.5%, or at least 99.8%, or at least99.9%. Homology can be determined using software programs readilyavailable in the art, such as those discussed in Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30,section 7.718, Table 7.71. Some alignment programs are MacVector (OxfordMolecular Ltd, Oxford, U.K.) and ALIGN Plus (Scientific and EducationalSoftware, Pennsylvania). Other non-limiting alignment programs includeSequencher (Gene Codes, Ann Arbor, Mich.), AlignX, and Vector NTI(Invitrogen, Carlsbad, Calif.).

As used herein, the term “hybrid” refers to any individual cell, tissueor plant resulting from a cross between parents that differ in one ormore genes.

As used herein, the term “inbred” or “inbred line” refers to arelatively true-breeding strain.

The term “single allele converted plant” as used herein refers to thoseplants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered in addition tothe single allele transferred into the inbred via the backcrossingtechnique.

As used herein, the term “line” is used broadly to include, but is notlimited to, a group of plants vegetatively propagated from a singleparent plant, via tissue culture techniques or a group of inbred plantswhich are genetically very similar due to descent from a commonparent(s). A plant is said to “belong” to a particular line if it (a) isa primary transformant (TO) plant regenerated from material of thatline; (b) has a pedigree comprised of a TO plant of that line; or (c) isgenetically very similar due to common ancestry (e.g., via inbreeding orselfing). In this context, the term “pedigree” denotes the lineage of aplant, e.g. in terms of the sexual crosses affected such that a gene ora combination of genes, in heterozygous (hemizygous) or homozygouscondition, imparts a desired trait to the plant.

As used herein, the terms “introgression”, “introgressed” and“introgressing” refer to the process whereby genes of one species,variety or cultivar are moved into the genome of another species,variety or cultivar, by crossing those species. The crossing may benatural or artificial. The process may optionally be completed bybackcrossing to the recurrent parent, in which case introgression refersto infiltration of the genes of one species into the gene pool ofanother through repeated backcrossing of an interspecific hybrid withone of its parents. An introgression may also be described as aheterologous genetic material stably integrated in the genome of arecipient plant.

As used herein, the term “population” means a genetically homogeneous orheterogeneous collection of plants sharing a common genetic derivation.

As used herein, the term “variety” or “cultivar” means a group ofsimilar plants that by structural features and performance can beidentified from other varieties within the same species. The term“variety” as used herein has identical meaning to the correspondingdefinition in the International Convention for the Protection of NewVarieties of Plants (UPOV treaty), of Dec. 2, 1961, as Revised at Genevaon Nov. 10, 1972, on Oct. 23, 1978, and on Mar. 19, 1991. Thus,“variety” means a plant grouping within a single botanical taxon of thelowest known rank, which grouping, irrespective of whether theconditions for the grant of a breeder's right are fully met, can be i)defined by the expression of the characteristics resulting from a givengenotype or combination of genotypes, ii) distinguished from any otherplant grouping by the expression of at least one of the saidcharacteristics and iii) considered as a unit with regard to itssuitability for being propagated unchanged.

As used herein, the term “mass selection” refers to a form of selectionin which individual plants are selected and the next generationpropagated from the aggregate of their seeds. More details of massselection are described herein in the specification.

As used herein, the term “open pollination” refers to a plant populationthat is freely exposed to some gene flow, as opposed to a closed one inwhich there is an effective barrier to gene flow.

As used herein, the terms “open-pollinated population” or“open-pollinated variety” refer to plants normally capable of at leastsome cross-fertilization, selected to a standard, that may showvariation but that also have one or more genotypic or phenotypiccharacteristics by which the population or the variety can bedifferentiated from others. A hybrid, which has no barriers tocross-pollination, is an open-pollinated population or anopen-pollinated variety.

As used herein, the term “self-crossing”, “self pollinated” or“self-pollination” means the pollen of one flower on one plant isapplied (artificially or naturally) to the ovule (stigma) of the same ora different flower on the same plant.

As used herein, the term “cross”, “crossing”, “cross pollination” or“cross-breeding” refer to the process by which the pollen of one floweron one plant is applied (artificially or naturally) to the ovule(stigma) of a flower on another plant.

As used herein, the term “derived from” refers to the origin or source,and may include naturally occurring, recombinant, unpurified, orpurified molecules. A nucleic acid or an amino acid derived from anorigin or source may have all kinds of nucleotide changes or proteinmodification as defined elsewhere herein.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T and G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

A probe comprises an identifiable, isolated nucleic acid that recognizesa target nucleic acid sequence. A probe includes a nucleic acid that isattached to an addressable location, a detectable label or otherreporter molecule and that hybridizes to a target sequence. Typicallabels include radioactive isotopes, enzyme substrates, co-factors,ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.Methods for labelling and guidance in the choice of labels appropriatefor various purposes are discussed, for example, in Sambrook et al.(ed.), Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 andAusubel et al. Short Protocols in Molecular Biology, 4^(th) ed., JohnWiley & Sons, Inc., 1999.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; Ausubel et al. Short Protocols inMolecular Biology, 4^(th) ed., John Wiley & Sons, Inc., 1999; and Inniset al. PCR Protocols, A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif., 1990. Amplification primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as PRIMER (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). One of ordinaryskill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, in order to obtaingreater specificity, probes and primers can be selected that comprise atleast 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of atarget nucleotide sequences.

For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (2001) Molecular Cloning: A Laboratory Manual (3rd ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

The present disclosure provides an isolated nucleic acid sequencecomprising a sequence selected from the group consisting of FusR1,homologs of FusR1, orthologs of FusR1, paralogs of FusR1, and fragmentsand variations thereof. In one embodiment, the present disclosureprovides an isolated polynucleotide encoding a protein produced by thenucleic acid sequence for FusR1, comprising a nucleic acid sequence thatshares at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%identity to FusR1.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math., 2:482, 1981); Needleman and Wunsch (J.Mol. Biol., 48:443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci.,85:2444, 1988); Higgins and Sharp (Gene, 73:237-44, 1988); Higgins andSharp (CABIOS, 5:151-53, 1989); Corpet et al. (Nuc. Acids Res.,16:10881-90, 1988); Huang et al. (Comp. Appls Biosci., 8:155-65, 1992);and Pearson et al. (Meth. Mol. Biol., 24:307-31, 1994). Altschul et al.(Nature Genet., 6:119-29, 1994) presents a detailed consideration ofsequence alignment methods and homology calculations.

The present disclosure also provides a chimeric gene comprising theisolated nucleic acid sequence of any one of the polynucleotidesdescribed above operably linked to suitable regulatory sequences.

The present disclosure also provides a recombinant construct comprisingthe chimeric gene as described above. In one embodiment, saidrecombinant construct is a gene silencing construct, such as used inRNAi gene silencing. In another embodiment, said recombinant constructis a gene editing construct, such as used in CRISPR-Cas gene editingsystem.

The expression vectors of the present disclosure may include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria.

The present disclosure also provides a transformed host cell comprisingthe chimeric gene as described above. In one embodiment, said host cellis selected from the group consisting of bacteria, yeasts, filamentousfungi, algae, animals, and plants including, but not limited to Musagenus.

These sequences allow the design of gene-specific primers and probes forFusR1, homologs of FusR1, orthologs of FusR1, homeologs of FusR1,paralogs of FusR1, and fragments and variations thereof.

II. Modulation of Disease Resistance

The present disclosure is drawn to polynucleotides and/or polypeptidesof newly-identified FusR1 ( Fusarium Resistant 1) and methods formodulating, stimulating or enhancing disease resistance in plants,caused by pathogens. Pathogens of the disclosure include, but are notlimited to, bacteria, fungi, viruses or viroids, nematodes, insects, andthe like.

Bacterial pathogens include but are not limited to Pseudomonas avenaesubsp. avenae, Xanthomonas campestris pv. holcicola, Enterobacterdissolvens, Envinia dissolvens, Ervinia carotovora subsp. carotovora,Envinia chrysanthemi pv. zeae, Pseudomonas andropogonis, Pseudomonassyringae pv. coronafaciens, Clavibacter michiganensis subsp.,Corynebacterium michiganense pv. nebraskense, Pseudomonas syringae pv.syringae, Herniparasitic bacteria (see under fungi), Bacillus subtilis,Envinia stewartii, and Spiroplasma kunkelii.

Fungal pathogens include but are not limited to Collelotrichumgraminicola, Glomerella graminicola Politis, Glomerella lucumanensis,Aspergillus flavus, Rhizoctonia solani Kuhn, Thanatephorus cucumeris,Acremonium strictum W. Gams, Cephalosporium acremonium Auct. non CordaBlack Lasiodiplodia theobromae=Bolr odiplodia y theobromas Borde blancoMarasmiellus sp., Physoderma maydis, Cephalosporium Corticium sasakii,Curvularia clavata, C. maculans, Cochhobolus eragrostidis, Curvulariainaequahs, C. intermedia (teleomorph Cochhobolus intermedius),Curvularia lunata (teleomorph: Cochliobolus lunatus), Curvulariapallescens (teleomorph Cochliobolus pallescens), Curvulariasenegalensis, C. luberculata (teleomorph: Cochliobolus tuberculatus),Didymella exitalis Diplodiaftumenti (teleomorph—Botryosphaeriafestucae),Diplodia maydis=Stenocarpella maydis, Stenocarpella macrospora=Diplodiamacrospora, Sclerophthora rayssiae var. zeae, Sclerophthoramacrospora=Sclerospora macrospora, Sclerospora graminicola,Peronosclerospora maydis=Sclerospora maydis, Peronosclerosporaphilippinensis, Sclerospora philippinensis, Peronosclerosporasorghi=Sclerospora sorghi, Peronosclerospora spontanea=Sclerosporaspontanea, Peronosclerospora sacchari=Sclerospora sacchari, Nigrosporaoryzae (teleomorph: Khuskia oryzae) A. Iternaria alternala=A. tenuis,Aspergillus glaucus, A. niger, Aspergillus spp., Botrytis cinerea,Cunninghamella sp., Curvulariapallescens, Doratomycesslemonitis=Cephalotrichum slemonitis, Fusarium culmorum, Gonatobotryssimplex, Pithomyces maydicus, Rhizopus microsporus Tiegh., R.stolonifer=R. nigricans, Scopulariopsis brumptii, Claviceps gigantea(anamorph: Sphacelia sp.) Aureobasidium zeae=Kabatiella zeae, Fusariumsubglutinans=F. moniliforme var. subglutinans, Fusarium moniliforme,Fusarium avenaceum (teleomorph Gibberella avenacea), Botryosphaeriazeae=Physalospora zeae (anamorph: Allacrophoma zeae), Cercosporasorghi=C. sorghi var. maydis, Helminthosporium pedicellatum (teleomorph:Selosphaeriapedicellata), Cladosporium cladosporioides=Hormodendrumcladosporioides, C. herbarum (teleomorph Mycosphaerella tassiana),Cephalosporium maydis, A. Iternaria alternata, A. scochyta maydis, A.tritici, A. zeicola, Bipolaris victoriae, Helminthosporium victoriae(teleomorph Cochhoholus victoriae), C. sativus (anamorph: Bipolarissorokiniana=H. sorokinianum=H. sativum), Epicoccum nigrum, Exserohilumprolatum=Drechslera prolata (teleomorph: Setosphaeriaprolata), Graphiumpenicillioides, Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerellaherpotricha (anamorph Scolecosporiella sp.), Pataphaeosphaeria michotii,Phoma sp., Septoria zeae, S. zeicola, S. zeina Setosphaeria turcica,Exserohilzim turcicum=Helminthosporium furcicum, Cochhoholus carbonum,Bipolaris zeicola=Helminthosporium carhonum, Penicilhum spp., P.chrysogenum, P. expansum, P. oxalicum, Phaeocytostroma ambiguum,Phaeocylosporella zeae, Phaeosphaeria maydis=Sphaerulina nmaydis,Botryosphaeriafestucae=Physalospora zeicola (anamorph:Diplodiaftumenfi), Herniparasitic bacteria and fungi Pyrenochaeta Phomaterrestris=Pyrenochaeta terrestris, Pythiumn spp., P. arrhenomanes, P.graminicola, Pythium aphanidermatum=P. hutleri L., Rhizoctonia zeae(teleomorph: Waitea circinata), Rhizoctonia solani, minor A Iternariaalternala, Cercospora sorghi, Dictochaetaftrtilis, Fusarium acuminatum(teleomorph Gihherella acuminata), E. equiseti (teleomorph: G.intricans), E. oxysporum, E. pallidoroseum, E. poae, E. roseum, G.cyanogena (anamorph: E. sulphureum), Microdochium holleyi, Mucor sp.,Periconia circinata, Phytophthora cactorum, P. drechsleri, P. nicotianaevar. parasitica, Phytophthora spp., Rhizopus arrhizus, Setosphaeriarostrata, Exserohilum rostratum=Helminthosporium rostratum, Pucciniasorghi, Physopella pallescens, P. zeae, Sclerotium rofsii Sacc.(teleomorph Athelia rotfsii), Bipolaris sorokiniana, B.zeicola=Helminthosporium carbonum, Diplodia maydis, Exserohilumpedicillatum, Exserohilum furcicum=Helminthosporium turcicum, Fusariumavenaceum, E. culmorum, E. moniliforme, Gibberella zeae (anamorph E.graminearum), Macrophominaphaseolina, Penicillium spp., Phomopsis sp.,Pythium spp., Rhizoctonia solani, R. zeae, Sclerotium rolfsfi, Spicariasp., Selenophoma sp., Gaeumannomyces graminis, Myrothecium gramineum,Monascus purpureus, M ruber Smut, Ustilago zeae=U. maydis Smut,Ustilaginoidea virens Smut, Sphacelotheca reiliana=Sporisorium holci,Cochliobolus heterostrophus (anamorph: Bipolaris maydis=Helminthosporiummaydis), Stenocarpella macrospora=Diplodia macrospora, Cercosporasorghi, Fusarium episphaeria, E. merismoides, F. oxysporum Schlechtend,Fusarium oxysporum f sp. cubense (Foc), Fusarium spp., E. poae, E.roseum, E. solani (teleomorph: Nectria haematococca), F. tricincturn,Mariannaea elegans, Mucor sp., Rhopographus zeae, Spicaria sp.,Aspergillus spp., Penicillium spp., Trichoderma viride=T. lignorumteleomorph: Hypocrea sp., Stenocarpella maydis=Diplodia zeae, Ascochytaischaemi, Phyllosticta maydis (teleomorph: Mycosphaerella zeae-maydis),Mycosphaerella fijiensis, Pseudocercospora (Paracercospora) fijiensi andGloeocercospora sorghi.

Virus or viroids include but are not limited to American wheat striatemosaic virus mosaic (AWSMV), barley stripe mosaic virus (BSMV), barleyyellow dwarf virus (BYDV), banana bunchy top virus, Brome mosaic virus(BMV), cereal chlorotic mottle virus (CCMV), corn chlorotic vein bandingvirus (CCVBV), maize chlorotic mottle virus (MCMV), maize dwarf mosaicvirus (MDMV), A or B, wheat streak mosaic virus (WSMV), cucumber mosaicvirus (CMV), cynodon chlorotic streak virus (CCSV), Johnsongrass mosaicvirus (JGMV), maize bushy stunt or mycoplasma-like organism (NILO),maize chlorotic dwarf virus (MCDV), maize chlorotic mottle virus (MCMV),maize dwarf mosaic virus (MDMV) strains A, D, E and F, maize leaf fleckvirus (MLFV), maize line virus (NELV), maize mosaic virus (MMV), maizemottle and chlorotic stunt virus, maize pellucid ringspot virus (MPRV),maize raya gruesa virus (MRGV), maize rayado fino virus (MRFV), maizered leaf and red stripe virus (MRSV), maize ring mottle virus (MRMV),maize rio cuarto virus (MRCV), maize rough dwarf virus (MRDV), maizesterile stunt virus (strains of barley yellow striate virus), maizestreak virus (MSV), maize chlorotic stripe, maize hoja Maize stripevirus blanca, maize stunting virus, maize tassel abortion virus (MTAV),maize vein enation virus (MVEV), maize wallaby ear virus (MAVEV), maizewhite leaf virus, maize white line mosaic virus (NTVVLMV), millet redleaf virus (NMV), viruses of the family Nanoviridae, Northern cerealmosaic virus (NCMV), oat pseudorosette virus, oat sterile dwarf virus(OSDV), rice black-streaked dwarf virus (RBSDV), rice stripe virus(RSV), sorghum mosaic virus (SrMV), formerly sugarcane mosaic virus(SCMV) stains H, I and M, sugarcane Fiji disease virus (FDV), sugarcanemosaic virus (SCMV) strains A, B, D, E, SC, BC, Sabi and NM vein enationvirus, and wheat spot mosaic virus (WSMV).

Parasitic nematodes include but are not limited to Awl Dolichodorusspp., D. heterocephalus Bulb and stem (Europe), Ditylenchus dipsaciBurrowing Radopholus similis Cyst Heterodera avenae, H. zeae, Punctoderachalcoensis Dagger Xiphinema spp., X. americanum, X. mediterraneum Falseroot-knot Nacobbus dorsalis Lance, Columbia Hoplolaimus columbus LanceHoplolaimus spp., H. galeatus Lesion Pratylenchus spp., P. brachyurus,P. crenalus, P. hexincisus, P. neglectus, P. penetrans, P. scribneri, P.thornei, P. zeae Needle Longidorus spp., L. breviannulatus RingCriconemella spp., C. ornata Root-knot Meloidogyne spp., M. chitwoodi,M. incognita, M. javanica Spiral Helicotylenchus spp., Belonolaimusspp., B. longicaudatus Stubby-root Paratrichodorus spp., P. christiei,P. minor, Ouinisulcius aculus, and Trichodorus spp.

Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Coleoptera and Lepidoptera.

In some embodiments, the plant pathogen is selected from fungi,especially soil borne fungi such as Fusarium oxysporum, water andair-borne viruses such as Mycosphaerella fijiensis (Morelet),Mycosphaerella musicola (Leach ex Mulder), Pseudocercospora(Paracercospora) fijiensi, Verticillium dahliae, Cladosporium andRalstona Solanaceum.

In some embodiments, said disease is Fusarium wilt, also known as Panamadisease, which is a lethal fungal disease caused by the soil-bornefungus Fusarium oxysporum f. sp. cubense (Foc). Said disease can also beknown as Panama Disease TR4, Foc, Panama Disease Tropical Race 4, orTR4. In some embodiments, resistance to TR4 is combined within a singlecultivar with genetic resistances or tolerances to one or moreadditional diseases, such as resistance to diseases caused by bacteria,other fungi, viruses, nematodes, insects and the like.

Fusarium wilt is one of the most destructive and notorious diseases ofbanana. It is also known as Panama disease, in recognition of theextensive damage it caused in export plantations in this CentralAmerican country. By 1960, Fusarium wilt had destroyed an estimated40,000 ha of ‘Gros Michel’ (AAA), causing the export industry to convertto cultivars in the Cavendish subgroup (AAA) (Ploetz and Pegg, 2000).Fusarium wilt is caused by the soil-borne hyphomycete, Fusariumoxysporum Schlect. f sp. cubense. It is one of more than 120 formaespeciales (special forms) of F. oxysporum that cause vascular wilts offlowering plants. This pathogen affects species of Musa and Heliconia,and strains have been classified into four physiological races based onpathogenicity to host cultivars in the field (race 1, ‘Gros Michel’;race 2, ‘Bluggoe’; race 3, Heliconia spp.; and race 4, Cavendishcultivars and all cultivars susceptible to race 1 and 2). Four Fusariumoxysporum races have been named, Race 1 through Race 4. Race 1 is acritical pathogen of many banana cultivars. Race 2 attacks cookingbananas. Race 3 affects banana relatives in the Americas, but doesn'tseem to affect bananas. The current threat stems from the expansion ofFusarium oxysporum race 4, also known as TR4 (Tropical Race 4), which isdesignated as ‘Foc-TR4’. Race 4 has two subgroups, TR4 and SR4(subtropical race 4). Until recently, race 4 had only been recorded tocause serious losses in the subtropical regions of Australia, SouthAfrica, the Canary Islands, and Taiwan. Banana growers and bananacompanies have repeatedly stated that if this race were to becomeestablished in the Americas, the world export industries would beseverely affected, as there is no widely accepted replacement forCavendish cultivars (Bentley et al., 1998).

Very recently, (Stokstad, 2019), Panama Disease Race 4 (Fusarium wilt)has now been detected in the Western Hemisphere. The disease was foundin four plantations in Columbia. These four plantations were immediatelyquarantined. However, a substantial part of the banana market consistsof exports from Central and South America to the United States. Thismarket is now critically imperiled, making a swift solution to thecrisis even more urgent. The recent emergence of Panama Disease TR4 inthe Western Hemisphere makes a swift solution to the crisis even moreurgent.

In some embodiments, ‘Fusarium Wilt” or ‘FW’ can be usedinterchangeably, which designates the disease as displayed in infectedbanana plants.

In the 1950s and 1960s, a single variety, Gros Michel, was grown widely.It was highly sensitive to the easily spread fungus Fusarium oxysporum fsp. cubense. In particular, it was Fusarium Tropical Race 1 (Foc-TR1)which caused a fatal wilt disease, and the global banana industry wasnearly destroyed. The Cavendish variety was found to be highly resistantto Foc-TR1, and replaced Gros Michel for global banana production. Inthe 1990s, growers began to find banana plants infected with Foc-TR4, anewly emerging race. Foc-TR4 is also easily spread and has been found inbanana plantations in Asia, the Middle East, and Africa, againthreatening the global banana crop. Great concern has been provoked bythe recent identification of Foc-TR4 in the Caribbean, which means thatthe fungus now has a beachhead in the Western Hemisphere, thusthreatening Latin America banana production. In some embodiments, thepresent disclosure provide a solution to serious problems on bananascaused by Foc-TR4. In some embodiments, the solution is drawn toidentification of disease-resistant genetic materials and/orarchitecture and importation of said genetic materials and architectureto banana varieties that are susceptible to pathogenic fungi (e.g.Foc-TR4).

Bananas are also susceptible to other pathogenic fungi, particularlyMycosphaerella fijiensis (Morelet) which causes black leaf streakdisease (also known as Black Sigatoka and Black Sig) and M. musicola,which causes Yellow Sigatoka leaf spot disease. It is known that thesefungi fijiensis and M. musicola) are controlled with fungicides, butfungicides are ineffective against Foc-TR4.

The present disclosure teaches method of modulating, stimulating, orenhancing disease resistance in plants, caused by pathogens such asFoc-TR4 using next generation plant breeding techniques, also known asnew breeding techniques.

New breeding techniques (NBTs) refer to various new technologiesdeveloped and/or used to create new characteristics in plants throughgenetic variation, the aim being targeted mutagenesis, targetedintroduction of new genes or gene silencing (RdDM). The followingbreeding techniques are within the scope of NBTs: targeted sequencechanges facilitated through the use of Zinc finger nuclease (ZFN)technology (ZFN-1, ZFN-2 and ZFN-3, see U.S. Pat. No. 9,145,565,incorporated by reference in its entirety), Oligonucleotide directedmutagenesis (ODM, a.k.a., site-directed mutagenesis), Cisgenesis andintragenesis, epigenetic approaches such as RNA-dependent DNAmethylation (RdDM, which does not necessarily change nucleotide sequencebut can change the biological activity of the sequence), Grafting (on GMrootstock), Reverse breeding, Agro-infiltration for transient geneexpression (agro-infiltration “sensu stricto”, agro-inoculation, floraldip), Transcription Activator-Like Effector Nucleases (TALEN5, see U.S.Pat. Nos. 8,586,363 and 9,181,535, incorporated by reference in theirentireties), the CRISPR/Cas system (see U.S. Pat. Nos. 8,697,359;8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308;8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641, which are allhereby incorporated by reference), engineered meganuclease,re-engineered homing endonucleases, DNA guided genome editing (Gao etal., Nature Biotechnology (2016), doi: 10.1038/nbt.3547, incorporated byreference in its entirety), and Synthetic genomics. A major part oftoday's targeted genome editing, another designation for New BreedingTechniques, is the applications to induce a DNA double strand break(DSB) at a selected location in the genome where the modification isintended. Directed repair of the DSB allows for targeted genome editing.Such applications can be utilized to generate mutations (e.g., targetedmutations or precise native gene editing) as well as precise insertionof genes (e.g., cisgenes, intragenes, or transgenes). The applicationsleading to mutations are often identified as site-directed nuclease(SDN) technology, such as SDN1, SDN2 and SDN3. For SDN1, the outcome isa targeted, non-specific genetic deletion mutation: the position of theDNA DSB is precisely selected, but the DNA repair by the host cell israndom and results in small nucleotide deletions, additions orsubstitutions. For SDN2, a SDN is used to generate a targeted DSB and aDNA repair template (a short DNA sequence identical to the targeted DSBDNA sequence except for one or a few nucleotide changes) is used torepair the DSB: this results in a targeted and predetermined pointmutation in the desired gene of interest. As to the SDN3, the SDN isused along with a DNA repair template that contains new DNA sequence(e.g. gene). The outcome of the technology would be the integration ofthat DNA sequence into the plant genome. The most likely applicationillustrating the use of SDN3 would be the insertion of cisgenic,intragenic, or transgenic expression cassettes at a selected genomelocation. A complete description of each of these techniques can befound in the report made by the Joint Research Center (JRC) Institutefor Prospective Technological Studies of the European Commission in 2011and titled “New plant breeding techniques—State-of-the-art and prospectsfor commercial development”, which is incorporated by reference in itsentirety.

In some embodiments, various approaches have been taken to prevent ortreat Foc-TR4 infection. The present disclosure teaches that a keyapproach to prevent or treat Foc-TR4 is to (1) find resistant bananacultivars, (2) to identify resistance genes and/or traits from theselected banana cultivars, and (3) breed/introduce the resistance genesand/or traits into sensitive banana cultivars.

Zuo et al. (2018) evaluated 129 banana accessions and found 10 that arehighly resistant to Foc-TR4—thus providing naturally existing resistantcultivars for study.

Li et al. (2012) looked at the transcriptomes and expression profiles ofroots of a resistant mutant and compared these to sensitive wild typeBrazilian Cavendish bananas at two time points after challenge withFoc-TR4. They found some 88,000 unigenes, with 5,000 related to defensepathways in other plants. They concluded that some 2,600 genes weredifferentially expressed in the resistant mutant, including some plantcell lignification genes that were expressed at the same or lower levelsin the resistant mutant.

In similar fashion, Bai et al. (2013) compared root transcriptomes fromthe Foc-TR4 sensitive Brazilian cultivar to the Yueyoukangl cultivarthat is known to have far lower disease severity. Bai et al. founddifferential expression for 500 to 2000 different unigenes at differenttime points, and these could be clustered into 11 different types ofmetabolic pathways. Bai et al. found genes connected to cell walllignification that were differentially regulated between the sensitiveand resistant cultivars—specifically 4-coumarate: CoA ligase (4CL),glutathione S-transferase (GST), cellulose synthase, Caffeoyl-CoAO-methyltransferase (CCoAM), and cinnamylalcohol dehydrogenase (CAD)were expressed at higher levels in the resistant cultivar and concludedthat cell wall lignification could be one of the mechanisms involved inFoc-TR4 resistance. Bai et al. pointed out that this was inconsistentwith the result found in Li et al. (2012) and concluded that differentplants could have different resistance mechanisms and that more work isrequired to decipher how banana cultivars are able to resist Foc-TR4.

Wang et al. (2017) also looked at differential root gene expression atthe time of flower bud differentiation and found 107 genesdifferentially expressed in the roots between a susceptible bananacultivar and a sensitive one.

Zhang et al. (2018) showed that Foc-TR4 infection proceeds similarly inthe roots of a resistant cultivar (Pahang) and a susceptible cultivar(Brazilian) until reaching the corm, where the fungal biomass and degreeof necrosis were significantly less in the Pahang vs. Brazilian. (Thebanana ‘corm’ is an underground stem, or rhizome, from which the rootsgrow.)

Van der Berg et al. (2007) used quantitative RT-PCR to identify genesthat are were up-regulated in the FW-tolerant GCTCV-218 banana cultivarafter infection with Foc-4. Their control was the FW-sensitive Williamscultivar. They found that a number of genes were up-regulated inFW-tolerant GCTCV-218 as compared to FW-sensitive Williams. As expected,many of the up-regulated genes were homologous to knowndefense-associated genes, including cell wall-strengthening genes. Theyreported 13 genes that were up-regulated in roots. While they state that“The results shed light on genes involved in defence and provide a steptowards understanding Fusarium wilt of banana and thereby developing aneffective disease management strategy”, the paper does not suggest thatany one of 13 that they deposited in GenBank can be used for controllingFusarium Wilt. No particular strategy is given for use of these genes tocontrol Fusarium Wilt.

Vishnevetsky et al. (2009) (U.S. Pat. No. 7,534,930) described a methodto genetically engineer banana plants to confer exogenous diseaseresistance traits, including resistance to Black and Yellow Sigatoka andBotrytis cinerea. Vishnevetsky et al. manipulated three polynucleotidesinto banana plants, including genes encoding endochitinase, stilbenesynthase, and superoxide dismutase.

Paul et al (2011) isolated a gene from the nematode C. elegans that,when stably transformed into the ‘Lady finger’ banana cultivar, appearedin greenhouse trials to confer resistance to Race 1 of Panama Disease.

Although transformation of bananas with a gene derived from a nematodeis unlikely to be accepted by consumers, follow-up work by Dale's groupwith a gene derived from bananas does show promise for achievingFusarium resistance in GMO-transformed bananas. For example,Peraza-Echeverria et al (2009) isolated a resistance gene analog (RGA2)gene from a wild banana, Musa acuminata malaccensis. This gene is amember of the large NB-LRR-type resistance gene family. When transformedinto FW-sensitive Cavendish plants (Dale et al, 2017), the gene appearsto confer resistance to Fusarium. Dale et al (2017) conducted fieldtrials of transgenic banana plants for 3 years. At the trial'sconclusion, some 67% to 100% of FW-sensitive control plants were dead orinfected. However, in four lines of bananas transfected with theircandidate gene, fewer than 30% of the transformed bananas showed signsof severe infection (i.e., >70% showed some tolerance or resistance).One line transformed with RGA2 appeared to be immune to TR4. While thisis good evidence that the gene may confer some FW-resistance, the genewas first isolated over a decade ago and it is unclear whether thebanana growing industry will ever embrace the RGA2 gene.

It is important to note that it is believed (unpublished communicationswith banana industry breeders and scientists) that there may be up tofour genes in the Musa genome that contribute some degree of Fusariumresistance so RGA2 alone is unlikely to solve the present crisis, evenif it is accepted by growers. Even if RGA2 finds acceptance, that theindustry has a dire need for multiple genes to control TR4.

Inventor notes that FusR1 of the present disclosure is completelyunrelated to RGA2. The two genes have completely different nucleotidesequences (i.e., they have no sequence identity), they lie on differentchromosomes, they have different biochemistries, and they have differentmechanisms of action in the plant.

Wu et al. (2016) sequenced a disease-resistant wild banana relative,Musa itinerans, found in subtropical China. Ks values were calculated inorder to estimate speciation and paleoploidization events in the Musagenus. Also Ka/Ks values were calculated to show that as expected, mostgenes in the Musa itinerans genome have undergone purifying selection.It was suggested that M. itinerans is known to be disease resistant,thus, its genome could be mined for disease resistance genes.

In some embodiments, the present disclosure provides methods of finding,identifying, and selecting genes resistant to diseases, such as Fusariumwilt from FW-resistant banana cultivars. In other embodiments, thepresent disclosure provides nucleotide and polypeptide sequences ofFusarium-resistant genes (e.g. FusR1 gene) identified from the methodsof the present disclosure. In further embodiments, the presentdisclosure teaches methods of generating and/or producing bananavarieties having resistance genes and/or traits by using next generationplant breeding technology, which include but are not limited to CRISPRtechnology described in the present disclosure.

III. Identification of FusR1 Gene from Musa Genus

Cultivated bananas are generally triploid (although a few are diploid)as a result of their complex evolutionary and domestication historywhich involved a number of interspecific and intraspecific hybridizationevents, both natural and human-driven. Edible, cultivated bananas arelargely the result of hybridization between two wild diploid species,Musa acuminata and Musa balbisiana (Christelová et al., 2017). Humandomestication of bananas began about 7,000 years ago in Southeast Asia(D'Hont et al, 2012). Banana genomes derived from M. acuminata are knownas “A” genomes, while bananas derived from M. balbisiana have “B”genomes (D'Hont et al., 2012). Thus the genome structure of the diploidM. acuminata is labeled AA, and the genomic structure of diploid M.balbisiana is BB. Edible banana cultivars may thus have triploid AAAgenomes (like Cavendish or Gros Michel), AAB genomes (as in manyplantains), or ABB genomes (like the Cachaco landrace). M. acuminatalikely arose in Malaysia or Indonesia (Christelova et al., 2017). Incontrast, M. balbisiana is believed to have originated in India,Thailand or the Philippines (Christelova et al., 2017). Thus, these twospecies were originally allopatric and geographic isolation provided anopportunity for each species to develop unique traits. When humans latermoved M. acuminata cultivars to areas populated by M. balbisiana,interspecific hybridization took place.

The economically critical Cavendish cultivar, which accounts for atleast 99% of commercial banana export production, exhibits triploidinduced sterility. This, combined with parthenocarpy, gives rise toedible fruit without seeds, but severely hampers breeding, so Cavendishbananas are propagated vegetatively (clonally). The Cavendish genotypehas three M. acuminata-derived “A” genomes.

In some embodiments, inventor identified genes that effectively controlFusarium Wilt in banana. For example, the present disclosure teachesthat a gene, which is named FusR1 ( Fusarium Resistance 1) wasidentified by using inventor's molecular evolutionary analysis approach.The resent disclosure teaches that the FusR1 gene is a native gene inMusa species, including cultivated bananas, M. itinerans, M. acuminata,M. balbisiana, M. basjoo, as well as Musella lasiocarpa, the sole memberof a closely related genus. The ortholog (two alleles) from the wildbanana relative, Musa itinerans, is given here as SEQ ID NO: 1 and SEQID NO: 4. The M. itinerans FusR1 sequences were obtained from multipleaccessions (including, but not limited to, ITC1526, ITC1571, andPT-BA-00223). All M. itinerans accessions are extremely FW-resistant (Liet al., 2015; Wu et al., 2016).

The present disclosure teaches that inventor identified two alleles ofFusR1 in M. itinerans. SEQ ID NO: 1 gives allele #1 of the FusR1 mRNAsequence. SEQ ID NO: 2 gives the allele #1 coding sequence. SEQ ID NO: 4gives allele #2 of the FusR1 mRNA sequence. SEQ ID NO: 5 gives theallele #2 coding sequence. Alleles 1 and 2 are very similar in sequence:they code for just four amino acid differences.

A second transcript of FusR1 was identified (SEQ ID NO: 7) from M.itinerans; this transcript has an expressed (i.e., unspliced) intronthat results in disruption of the proper reading frame. This isexpressed at very low levels.

M. itinerans is naturally extremely resistant to the effects of FusariumWilt (Li et al., 2015; Wu et al., 2016). In some embodiments, the FusR1gene from M. itinerans is responsible for resistance to Fusarium Wilt.

The present disclosure further teaches that inventor identified threealleles of FusR1 in M acuminata. Two of these alleles were isolated fromFW-resistant accessions of M. acuminata. The third allele was isolatedfrom an FW-sensitive M. acuminata accession. The M. acuminata FusR1FW-resistant sequences were obtained from multiple FW-resistantaccessions, including ITC0896 (M. a. subspecies banksii) and PT BA-00281(Pisang Bangkahulu). The M. acuminata FW-sensitive sequence is from theFW-sensitive accessions ITC0507, ITC0685, PT-BA-00304, PT-BA-00310, andPT-BA-00315.

SEQ ID NO: 8 gives the mRNA sequence of allele 1 of the FW-resistantFusR1 gene from M. acuminata. SEQ ID NO: 10 gives the mRNA sequence ofallele 2 of the FW-resistant FusR1 gene from M. acuminata. The codingsequence of FW-resistant allele 1 from M. acuminata is given in SEQ ID9. SEQ ID NO: 11 gives the coding sequence of FW-resistant allele 2 fromM. acuminata.

SEQ ID NO: 13 gives the mRNA sequence of the FW-sensitive FusR1 allelefrom M acuminata. (The M. acuminata FW-sensitive sequence was identifiedfrom accessions ITC0507, ITC0685, PT-BA-00304, PT-BA-00310, andPT-BA-00315. These accessions include multiple samples from bananacultivars such as Pisang Madu, Pisang Pipit, and Pisang Rojo Uter, allof which have been well-characterized as FW-sensitive (Chen et al,2019).

Inventor identified a putative core promoter for FusR1 from M.acuminata. Inventor used two different promoter prediction applicationsin an attempt to find congruent predictions from differentalgorithms/software.

As a first step, inventor amplified and sequenced a 753 bp sequencefragment (SEQ ID NO: 31), which begins upstream of the coding region ofthe FW-resistant-allele of the FusR1 gene derived from M. acuminata.This fragment is 100% identical to bp7868911-bp7869210 andbp7869341-bp7869743 of GenBank accession NC_025206 (Musa acuminatasubsp. malaccensis chromosome 5, ASM31385v2, whole genome shotgunsequence), which lies on M. acuminata Chromosome 5.

Inventor first analyzed the upstream region of FusR1 using the “NeuralNetwork Promoter Prediction” (NNPP), which is available on the BerkeleyDrosophila Genome Project (BDGP). BDGP is a consortium of the DrosophilaGenome Center, funded by the National Human Genome Research Institute,the National Cancer Institute, and the Howard Hughes Medical Institute.The NNPP software was ‘trained’ on human and Drosophila melanogasterpromoter sequences, but has proven to be generally effective atidentifying promoter sequences, even in plants (Reese, 2001).

NNPP analysis successfully identified a core promoter for FusR1.Analysis results follow. The first 189 bases of SEQ ID NO: 31 (shown inlower case) are non-coding upstream sequence, including the 5′ UTRsequence of FusR1; the next 423 bases are coding sequence (shown inUPPER CASE). This coding sequence is identical to SEQ ID NO: 9. The last141 bases are 3′ UTR (shown in lower case). Bases 92-141 of SEQ ID NO:31 (atcgtggcactataaataggacaagaggagggatgaggtaaaacgcactc) are the NNPPpredicted promoter sequence, shown in lower case bold. The transcriptionstart site (TSS) at base pair 132 is shown in lower case underlinedbold. NNPP assigns a score of 0.88 (i.e., 88% confidence level) to thispromoter.

SEQ ID NO: 31: gtagagacacttgagttgaattctgaatccattatttcttctcatgaacgcatacgtcccaccatacacaccaaatcttaatggctcaagcatcgtggca c

taggacaagaggagggatgaggta a aacgcactccctcatacttgcacaggtacgttgtgatagaaagttcagaggtaagcgATGGCTGGAGGAGGCAAAAGAGGTGAAGCGTCGTCTCTTCTACTTGTGACGCTGCTCGTGACGTTGTTGGCTTTCTTCGCCACCAACTCCTCGGCAGCCCGTGTCACACCCCGTCCGCAATCCCTCGCCAGAGCGGCACTGAGTGCGGTGGGGGCAAGGCAAGATGAGCCGTGCTGCAGATGCGCGTGTCCTCTCATTTACCCACCTACTTGGTGCATTTGCGGCGGCATATGGCAAGGCTCCTGCCCTTCCGCCTGCAACAACTGCCAGTGTGTCCTCAACGAGTGCACTTGCCTCGATCTTATGGACCCCAAGGTCTGCGAGGCCAACTCCTGTCCCTGGCCTGTTGCAGCCCCCAAAGTAGAGCCGGCGCAGCAGTGGGCTATCGAAGAAACCGGTGGGAAATTAGCGATGATGGTGTGAtccaattgtgtttgtgatcgcctgtcgtcttctctcgctccgtcctatccatctatccatccatctacttataatctatgtcgtgtaccgtcgtgtggtgttgctttgcttcagtaataaaaataaaatgcttctgct ttt

Inventor then analyzed the upstream region of FusR1 from M. acuminatausing the “Prediction of PLANT Promoters” (TSSP) software, which istargeted specifically at identification of plant promoter sequences(Solovyev and Shahmuradov, 2003). This is a part of a suite of sequenceanalysis software produced by Softberry, Inc. TSSP identified thetranscription start site (TSS) as position 132 in SEQ ID NO: 31, whichis identical to the NNPP software results (see above). TSSP located theFusR1 TATA box (shown above in lower case italics) at bases 102-107 ofSEQ ID NO: 31. Thus the FusR1 TATA box lies, as expected, 25 base pairsupstream of the TSS.

As these 2 different promoter prediction applications give congruentresults, inventor identified the correct promoter sequence for M.acuminata.

The present disclosure teaches methods of introducing thenewly-identified FusR1 gene and its variants into cultivated bananas,particularly the Fusarium-sensitive Cavendish cultivar in order to makethese cultivars resistant to Fusarium Wilt. In some embodiments, thepresent disclosure teaches that traditional plant breeding methods canbe used to introduce FusR1 gene/trait from M. itinerans into Cavendishand other cultivated bananas. In other embodiments, the presentdisclosure teaches that next generation plant breeding methods can beused to introduce FusR1 gene/trait from M. itinerans into Cavendish andother cultivated bananas. In further embodiments, the present disclosureteaches methods of introducing FusR1 gene/trait from M itinerans intoCavendish and other cultivated bananas using genome editing techniquessuch as targeted genome editing system using zinc finger nucleases(ZFN), transcription activator like effector nucleases (TALEN) orCRISPR/Cas9 system technology exploiting the endonuclease activity ofCRISPR-associated (Cas) proteins with sequence specificity directed byCRISPR RNAs (crRNAs).

Given the threat of likely extinction for Cavendish, the presentdisclosure provides a rapid, efficient, and precise genome editingapproach using CRISPR/Cas9 system adapted for production of minimallygenetically-edited bananas having Fusarium-resistant gene/trait, whichwill be accepted especially in developing countries where bananaprovides critical economic and food security. The present disclosureteaches that the transfer of the native FusR1 gene from M itinerans tocultivated bananas can be best accomplished with CRISPR technology,which allows a targeted, clean, and efficient transfer and which, ascompared to more traditional genetic editing techniques, minimizespotential side effects.

In some embodiments, useful alleles of FusR1 (SEQ ID NO: 8 and SEQ IDNO: 10) are identified from naturally FW-resistant M. acuminatapopulations. These alleles confer FW-resistance. The present disclosureteaches that the FusR1 allele derived from M. acuminata can be used, incombination with FusR1 alleles derived from M. itinerans (SEQ ID NO: 2and SEQ ID NO: 5), to enhance FW-resistance in cultivated bananas,particularly Cavendish.

The present disclosure teaches gene stacking with at least two FusR1genes identified by inventor disclosed in the present disclosure.

Both the M. itinerans FusR1 ortholog (SEQ ID NO: 2 and SEQ ID NO: 5) andthe M acuminata FW-resistant alleles (SEQ ID NO: 8 and SEQ ID NO: 10)can be used in traditional plant breeding and/or new generation plantbreeding approaches. The new generation plant breeding approachesinclude but are not limited to marker-assisted-selection (MAS) and/orgenome editing techniques in cultivated bananas.

Some M. balbisiana accessions have been rigorously characterized as veryresistant to FW, while others are extremely FW-sensitive. While it mightbe expected that the wild M balbisiana accessions would be resistant toa pathogen like Fusarium, it has been difficult for researchers tounderstand why closely related accessions differ so significantly interms of FW resistance.

Inventor discovered a structural difference of the nucleotide sequencesof FusR1 gene in FW-sensitive M. balbisiana accessions as shown in FIG.5. All the FW-sensitive M. balbisiana accessions inventor analyzedcontain a ‘broken’ FusR1 transcript. This analysis is restricted to the‘broken’ FusR1 genes found in all FW-sensitive accessions that wereexamined. FusR1 mRNAs in all M. balbisiana accessions inventor examinedhad an unspliced, expressed intron that disrupts proper reading frame.In addition, inventor found (i) a long 82 or 84 bp deletion in severalFusR1 mRNAs(2) in all accessions, a smaller 1 bp deletion, or (ii), insome accessions, a 4 bp insertion, each of which also disrupts the openreading frame, thus coding for a mutated, non-functional FUSR1 protein.All FW-sensitive M. balbisiana accessions have one or more of thesereading frame disrupters described above, resulting in a non-functionalprotein. In some embodiments, the present disclosure teaches that someM. balbisiana accessions have all four reading frame disrupters. SeeFIG. 5.

In other embodiments, inventor also discovered another significantdifference when studying FW-resistant vs. FW-sensitive M. acuminataaccessions. In some embodiments, FusR1 in M. acuminata give resistancevs. sensitivity depending on FusR1 alleles. The present disclosureteaches that two alleles, which turned out to be “resistant alleles”confer FW-resistance; SEQ ID NO: 8 and SEQ ID NO: 10. These two allelesare very similar in sequence to the FusR1 ortholog derived from theFW-resistant wild banana spaces, M. basjoo (SEQ ID NO: 17 and SEQ ID NO:20. The third allele, the FW-sensitive allele, is found only inFW-sensitive M. acuminata accessions (SEQ ID NO: 13).

The M. balbisiana FusR1 sequence (SEQ ID NO: 26 and SEQ ID NO: 27) doesnot confer FW resistance, because this gene is damaged (as it is in allthe FW-sensitive M. balbisiana accessions examined) by reading-framedisrupting indels and/or expressed unspliced introns that cause loss ofFW resistance.

In further embodiments, FusR1 sequences derived from FW-resistant M.acuminata accessions (SEQ ID NO: 8 and SEQ ID NO: 10) have a very highsequence similarity to the FusR1 ortholog derived from M. basjoo (SEQ IDID: 17). M. basjoo is a wild banana species that is very resistant to FW(Li et al., 2015). In other embodiments, the FusR1 sequence (SEQ ID NO:13) from FW-sensitive M. acuminata accessions differs from theFW-resistant M. acuminata alleles (SEQ ID NO: 8 and SEQ ID NO: 10).

The present disclosure teaches that FW-resistance in M. acuminatadepends upon having the allele found only in FW-resistant accessions.Although M. acuminata and M. balbisiana are more closely related to eachother than either is to M. itinerans or M. basjoo, the FusR1 sequencesthat control FW-resistance cluster together in direct contrast to theway the species are actually related. In other embodiments, the FusR1gene has adapted (i.e., been positively selected) so that FusR1 fails toreflect the actual relationships within Musa species. The presentdisclosure teaches two independent adaptive events (convergentevolution) or perhaps the FW-resistant FusR1 version has been tradedbetween various Musa species (gene transfer).

Inventor confirmed the true phylogenetic relationships between theseMusa species by sequencing two different, conserved, single-copy genes,C2H2 and TOPO6, from several Musa species. C2H2-type zinc fingerproteins play important roles in plant development and growth as well asabiotic stress resistance, including for fruit ripening in banana (Hanet al., Front. Plant Sci., Vol. 11, Article 115:1-13, 20 Feb. 2020; Hanet at, Postharvest Biology and Technology, 1.16:8-15, June 2010. TOPO6,a nuclear gene-marker region of subunit B of the plant homolog ofarchaean topoisomerase VI, occurs as single-copy locus in the haploidgenome of most plant groups (Frank R. Blattner, Plant Systematics andEvolution, Vol. 302: 239-244, 2016). These two genes (whose biochemicalfunctions are well-known) have no role in pathogen control, making themideal as ‘controls’ for understanding the adaptive changes imposed onbanana FusR1 as a result of exposure to Fusarium. Thus, the disclosureteaches that the consensus in the literature that M. acuminata and M.balbisiana are sister species is correct, meaning that significantchanges have occurred to our newly-identified gene, FusR1, in thesebanana species, providing yet more evidence that FusR1 confersFW-resistance. See the phylogenetic trees provided in FIGS. 3 and 4.

The present disclosure teaches the critical sequence differences betweenthe strongly FW-resistant FusR1 alleles from M. itinerans, which allowsthe inventor to determine the exact few nucleotides that make FusR1capable of controlling FW. Based on the inventor's findings, the presentdisclosure teaches a method of using CRISPR/Cas system to conferFW-resistance in FW-sensitive Cavendish (as well as all other cultivatedbananas), by precisely changing only a few critical nucleotides inFusR1. Also, the present disclosure also teaches a method of using thesecritical nucleotides to create a novel FusR1 sequence with greaterFW-resistance than the native gene.

IV. FusR1 Gene and Variants Thereof

The present disclosure is predicated, in part, on the isolation of novelFusR1 gene from banana varieties and species. The nucleotide sequencesof this FusR1 gene and its orthologs sequences are presented in SEQ IDNO: 1-2, 4-5, 7-11, 13-14, 16-18, 20-21, 23-24, and 26-31 respectively.

In some embodiments, SEQ ID NO: 1 is partial mRNA sequence for allele 1of FusR1 from Musa itinerans, the most Fusarium-resistant wild bananaspecies. SEQ ID NO: 4 is partial mRNA sequence for allele 2 of FusR1from Musa itinerans.

The aforementioned FusR1 alleles from M. itinerans (SEQ ID NO: 1 and SEQID NO: 4) code for slightly different proteins, which are SEQ ID NO: 3and SEQ ID NO: 6, respectively. The translated polypeptide of SEQ ID NO:1 is presented as SEQ ID NO: 3. The translated polypeptide of SEQ ID NO:4 is presented as SEQ ID NO: 6. These are only slightly different, withthe few differing amino acid residues all being biochemicallyconservative. In some embodiments, 5 different M. itinerans accessionswere sequenced and all accessions had these same two FusR1 alleles.

In some embodiments, SEQ ID NO: 8 and SEQ ID NO: 10 are partial mRNAs(including the full coding sequences). These are the FW-resistantalleles of FusR1 from Musa acuminata ssp. banksia (Accession No.ITC0896) and PT_BA-00281(Pisang Bankahulu). These two alleles differ ata single silent site. In other embodiments, SEQ ID NO: 13 represents theFW-sensitive allele from M. acuminata. In further embodiments, SEQ IDNO: 9 and SEQ ID NO: 11 represent the coding sequence for theFW-resistant alleles from M. acuminata. Also, SEQ ID NO: 12 representsthe FW-resistant protein sequence from M. acuminata, which is atranslated polypeptide sequence of SEQ ID NO: 8 and SEQ ID NO: 10.

In some embodiments, SEQ ID NO: 17 and SEQ ID NO: 20 are partial mRNAFusR1 allele sequences from M. basjoo, a wild banana species that isresistant to Fusarium. In other embodiments, SEQ ID NO: 23 is the FusR1sequence from another wild banana relative, Musella lasiocarpa.

It is noted that all of the mRNA sequences inventor reports herein aretechnically partial, as they lack a bit of 5′UTR and usually a few basesof the extreme end of the 3′UTR. The vast majority of the mRNAs reportedherein are very close to being full sequence.

In some embodiments, SEQ ID NO: 26, and SEQ ID NO: 28-30 are the partialmRNA FusR1 sequences from several different M. balbisiana accessions.SEQ ID NO: 27 is the FusR1 coding sequence from M. balbisiana. In someembodiments, a large number of FW-sensitive M balbisiana accessions wereexamined. In all the FusR1 sequences from FW-sensitive M balbisianaaccessions, the structure of the FusR1 sequence is broken and/ordamaged. All the FW-sensitive M. balbisiana accessions had a FusR1coding sequence with a 1 bp deletion at position 340 in the codingsequence. All FW-sensitive M. balbisiana accessions also had a longunspliced, expressed intron in the coding sequence. Several also had along (82-84 bp) deletion, some had another 4 bp deletion, and in allcases, a one base pair deletion (relative to FusR1 from other plantspecies, including all other banana accessions While it is true that 84bp, as a multiple of three, doesn't disrupt the reading frame, it doesremove 28 amino acid residues from the protein's primary structure, thuspotentially disrupting the folded protein's tertiary structure and thusnegatively impacting function. In any case, based on our findings, theubiquitous 1 bp deletion always results in reading frame disruption.

Inventor included mRNA sequences from Musa balbisiana accessions fromwhich inventor sequenced FusR1. These illustrate the various ways inwhich FusR1 is ‘broken’ in M balbisiana. Inventor notes herein thatEVERY M. balbisiana accession inventor analyzed has a broken FusR1 mRNAtranscript. FIG. 5 shows these M. balbisiana FusR1 sequences aligned.

M. balbisiana accession ITC1016 (SEQ ID NO: 26) contains an 82 base pairunspliced, expressed intron. This intron disrupts the reading frame,resulting in a premature termination codon located 8 bp into the intron,which causes a truncated 141 bp coding sequence (as opposed to theproper 423 bp coding sequence). In addition, this accession (and, infact, all M. balbisiana accessions) also has a one base pair deletion,located about 90 bp 5′-ward of the true termination codon, which (evenif the intron had been properly spliced out) results in a premature stopcodon, giving a truncated coding sequence.

M. balbisiana accession ITC0545 (SEQ ID NO: 28) contains the same 82base pair unspliced, expressed intron. This intron disrupts the readingframe, resulting in a premature stop codon located 8 bp into the intron,causing a truncated 141 bp coding sequence (as opposed to the proper 423bp coding sequence). Another 27 bp downstream of the expressed intronlies an 85 bp deletion. While this in combination with the 84 bpexpressed intron would mathematically restore the correct reading frame,(85 bp−82 bp=3 bp), as explained above, it causes the loss of 28 aminoacid residues that lie in a functionally critical region of the foldedFusR1 protein. In addition, this accession also has the one base pairdeletion, located about 90 bp 5′-ward of the true termination codon,which (even if the intron had been properly spliced out) results in apremature stop codon, giving a truncated coding sequence. Finally, theFusR1 mRNA from this accession also has a frame-disrupting 4 bpinsertion farther downstream.

M. balbisiana accession ITC0080 (SEQ ID NO: 29) contains the sameunspliced, expressed intron as the previous accessions, except that thisversion of the unspliced intron is 84 bp in length. While this expressedintron doesn't disrupt reading frame, it does introduce 28 extra aminoacid residues that lie in a functionally critical region of the foldedprotein and thus very likely prevents proper folding of the FusR1protein. In addition, this accession also has the one base pairdeletion, located about 90 bp 5′-ward of the true termination codon,which (even if the intron had been properly spliced out) results in apremature stop codon, giving a truncated coding sequence.

M. balbisiana accession ITC1527 (SEQ ID NO: 30) contains the sameunspliced, expressed intron as the previous accessions, this time 82 bplong. Again, this intron disrupts the reading frame, resulting in apremature stop codon located 8 bp into the intron, causing a truncated141 bp coding sequence (as opposed to the proper 423 bp codingsequence). In addition, the FusR1 mRNA from this accession has a 4 bpinsertion farther downstream. In addition, this accession also has theone base pair deletion, located about 90 bp 5′-ward of the truetermination codon, which (even if the intron had been properly splicedout) results in a premature stop codon, giving a truncated codingsequence.

All M. balbisiana accessions inventor analyzed have some combination ofone or more of these various flaws in their FusR1 mRNA.

Table 1 summarizes sequence information of the present disclosure.

TABLE 1 Summary of Sequence Information SEQ ID NO. Sequence Type OriginBrief Description SEQ ID Nucleotide Musa itinerans Partial mRNA sequencefor the NO: 1 FW*-resistant FusR1 transcript 1, allele 1 from Musaitinerans SEQ ID Nucleotide Musa itinerans FusR1 allele 1 FW-resistantNO: 2 coding sequence from M. itinerans SEQ ID Protein Musa itineransProtein sequence of FUSR1 FW- NO: 3 resistant allele 1 from M. itineransSEQ ID Nucleotide Musa itinerans Partial mRNA sequence for NO: 4FusR1transcript 1 FW-resistant allele 2 from Musa itinerans SEQ IDNucleotide Musa itinerans FusR1 FW-resistant allele 2 NO: 5 codingsequence from M. itinerans SEQ ID Protein Musa itinerans Proteinsequence of FUSR1 FW- NO: 6 resistant allele 2 from M. itinerans SEQ IDNucleotide Musa itinerans Partial mRNA sequence for NO: 7 FusR1transcript 2 from Musa itinerans SEQ ID Nucleotide Musa acuminata ssp.Partial mRNA sequence for FW- NO: 8 banksii resistant FusR1 allele 1from M. acuminata SEQ ID Nucleotide Musa acuminata ssp. Coding sequenceof FW- NO: 9 banksii resistant FusR1 allele 1 from M. acuminata SEQ IDNucleotide Musa acuminata ssp. Partial mRNA sequence for FW- NO: 10banksii resistant FusR1 allele 2 from M. acuminata SEQ ID NucleotideMusa acuminata ssp. Coding sequence of FW- NO: 11 banksii resistantFusR1 allele 2 from M. acuminata SEQ ID Protein Musa acuminata ssp.Protein sequence of FW- NO: 12 banksii resistant FUSR1 from M. acuminataSEQ ID Nucleotide Musa acuminata Partial mRNA sequence for NO: 13FW-sensitive FusR1 allele from M. acuminata SEQ ID Nucleotide Musaacuminata Coding sequence of FW- NO: 14 sensitive FusR1 allele from M.acuminata SEQ ID Protein Musa acuminata Protein sequence of FW-sensitiveNO: 15 FusR1 from M. acuminata SEQ ID Nucleotide Musa acuminata PartialmRNA sequence of FW- NO: 16 sensitive FusR1 transcript 2 from M.acuminata SEQ ID Nucleotide Musa basjoo Partial mRNA sequence of FusR1NO: 17 FW-resistant allele 1 from M. basjoo SEQ ID Nucleotide Musabasjoo Coding sequence of FusR1 FW- NO: 18 resistant allele 1 from M.basjoo SEQ ID Protein Musa basjoo Protein sequence of FusR1 FW- NO: 19resistant allele 1 from Musa basjoo SEQ ID Nucleotide Musa basjooPartial mRNA sequence of FW- NO: 20 resistant allele 2 of FusR1 from M.basjoo SEQ ID Nucleotide M. basjoo Partial coding sequence of FusR1 NO:21 FW-resistant allele 2 from M. basjoo SEQ ID Protein M. basjoo Partialprotein sequence of FW- NO: 22 resistant allele 2 of FusR1 from M.basjoo SEQ ID Nucleotide Musella lasiocarpa Partial mRNA sequence ofFusR1 NO: 23 from Musella lasiocarpa SEQ ID Nucleotide Musellalasiocarpa Coding sequence of FusR1 from NO: 24 M. lasiocarpa SEQ IDProtein Musella lasiocarpa Protein sequence of FUSR1 from NO: 25 M.lasiocarpa SEQ ID Nucleotide M. balbisiana Partial mRNA sequence ofFusR1 NO: 26 from M. balbisiana Accession ITC1016 SEQ ID Nucleotide M.balbisiana “Hypothetical” coding sequence NO: 27 from M. balbisianaAccession ITC1016 SEQ ID Nucleotide M. balbisiana Partial mRNA sequenceof FusR1 NO: 28 from M. balbisiana Accession ITC0545 SEQ ID NucleotideM. balbisiana Partial mRNA sequence of FusR1 NO: 29 from M. balbisianaAccession ITC0080 SEQ ID Nucleotide M. balbisiana Partial mRNA sequenceof FusR1 from NO: 30 M. balbisiana Accession ITC1527 SEQ ID NucleotideM. acuminata ssp. Upstream Sequence, including NO: 31 banksii promotersequence, of the FW- resistant allele 1 of FusR1 from M. acuminata SEQID Protein M. balbisiana Protein sequence of FUSR1 from NO: 32 M.balbisiana *FW—Fusarium wilt

In accordance with the present disclosure, the novel FusR1 gene and itsorthologs will be useful for facilitating the construction of cropplants that are resistant to pathogenic disease, especially diseasecaused by fungal pathogens, viruses, nematodes, insects and the like.The FusR1 genes of the present disclosure can also be used as markers ingenetic mapping as well as in assessing disease resistance in a plant ofinterest. Thus, the sequences can be used in breeding programs. See, forexample, Gentzbittel et al. (1998, Theor. Appl. Genet. 96:519-523).Additional uses for the sequences of the disclosure include using thesequences as bait to isolate other signaling components ondefense/resistance pathways and to isolate the corresponding promotersequences. The sequences may also be used to modulate plant developmentprocesses, such as pollen development, regulation of organ shape,differentiation of aleurone and shoot epidermis, embryogenic competence,and cell/cell interactions. See, generally, Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). The sequences of the presentdisclosure can also be used to generate variants (e.g., by ‘domainswapping’) for the generation of new resistance specificities.

The disclosure encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or “purified” nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the nucleic acid molecule or protein as foundin its naturally occurring environment. Thus, an isolated or purifiedpolynucleotide or polypeptide is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Suitably, an “isolated” polynucleotide is freeof sequences (especially protein encoding sequences) that naturallyflank the polynucleotide (i.e., sequences located at the 5′ and 3′ endsof the polynucleotide) in the genomic DNA of the organism from which thepolynucleotide was derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide was derived. A polypeptide that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. Whenthe protein of the disclosure or biologically active portion thereof isrecombinantly produced, culture medium suitably represents less thanabout 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

A portion of a FusR1 nucleotide sequence that encodes a biologicallyactive portion of a FusR1 polypeptide of the disclosure will encode atleast about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 400,500, 600, 700, 800, 900 or 1000 contiguous amino acid residues, oralmost up to the total number of amino acids present in a full-lengthFUSR1 polypeptide of the disclosure (for example, 140 amino acidresidues for SEQ ID NO: 3, 6, 12, 19, or 22, respectively). Portions ofa FusR1 nucleotide sequence that are useful as hybridization probes orPCR primers generally need not encode a biologically active portion of aFUSR1 polypeptide.

Thus, a portion of a FusR1 nucleotide sequence may encode a biologicallyactive portion of a FUSR1 polypeptide, or it may be a fragment that canbe used as a hybridization probe or PCR primer using standard methodsknown in the art. A biologically active portion of a FUSR1 polypeptidecan be prepared by isolating a portion of one of the FusR1 nucleotidesequences of the disclosure, expressing the encoded portion of the FUSR1polypeptide (e.g., by recombinant expression in vitro), and assessingthe activity of the encoded portion of the FUSR1 polypeptide. Nucleicacid molecules that are portions of an FusR1 nucleotide sequencecomprise at least about 15, 16, 17, 18, 19, 20, 25, 30, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides, oralmost up to the number of nucleotides present in a full-length FusR1nucleotide sequence disclosed herein (for example, about from 350 to 650nucleotides for SEQ ID NO: 1-2, 4-5, 8-10, 17-18, or 20-21,respectively).

The disclosure also contemplates variants of the disclosed nucleotidesequences. Nucleic acid variants can be naturally occurring, such asallelic variants (same locus), homologues (different locus), andorthologues (different organism) or can be non-naturally occurring.Naturally occurring variants such as these can be identified with theuse of well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as known inthe art. Non-naturally occurring variants can be made by mutagenesistechniques, including those applied to polynucleotides, cells, ororganisms. The variants can contain nucleotide substitutions, deletions,inversions and insertions. Variation can occur in either or both thecoding and non-coding regions. The variations can produce bothconservative and non-conservative amino acid substitutions (as comparedin the encoded product). For nucleotide sequences, conservative variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the amino acid sequence of one of the FUSR1 polypeptides ofthe disclosure. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis but which still encode a FUSR1polypeptide of the disclosure. Generally, variants of a particularnucleotide sequence of the disclosure will have at least about 30%, 40%50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%,desirably about 90% to 95% or more, and more suitably about 98% or moresequence identity to that particular nucleotide sequence as determinedby sequence alignment programs described elsewhere herein using defaultparameters.

Variant nucleotide sequences also encompass sequences derived from amutagenic or recombinant procedures such as ‘DNA shuffling’ which can beused for swapping domains in a polypeptide of interest with domains ofother polypeptides. With DNA shuffling, one or more different FusR1coding sequences can be manipulated to create a new FusR1 sequencepossessing desired properties. In this procedure, libraries ofrecombinant polynucleotides are generated from a population of relatedpolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. For example, using this approach, sequence motifs encoding adomain of interest may be shuffled between the FusR1 gene of thedisclosure and other known FusR1genes to obtain a new gene coding for aprotein with an improved property of interest, such broadening spectrumof disease resistance. Strategies for DNA shuffling are known in theart. See, for example: Stemmer (1994, Proc. Natl. Acad. Sci. USA91:10747-10751; 1994, Nature 370:389-391); Crameri et al. (1997, NatureBiotech. 15:436-438); Moore et al. (1997, J. Mol. Biol. 272:336-347);Zlang et al. (1997 Proc. Natl. Acad. Sci. USA 94:450-44509); Crameri etal. (1998, Nature 391:288-291); and U.S. Pat. Nos. 5,605,793 and5,837,458.

The present disclosure provides nucleotide sequences comprising at leasta portion of the isolated proteins encoded by nucleotide sequences forFusR1, homologs of FusR1, orthologs of FusR1, paralogs of FusR1, andfragments and variations thereof.

In some embodiments, the present disclosure provides a nucleotidesequence encoding FUSR1, and/or functional fragments and variationsthereof comprising a nucleotide sequence that shares at least about 70%,about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, or about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%,about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9%sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 17, or SEQ ID NO: 18. In some embodiments, a nucleotide sequenceencoding FUSR1 has the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 17, or SEQ ID NO: 18.

In some embodiments, the present disclosure provides nucleotidesequences for FusR1, homologs of FusR1, orthologs of FusR1, paralogs ofFusR1, and fragments and variations thereof comprising nucleotidesequences that share at least about 70%, about 75%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99%, about99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%,about 99.7%, about 99.8%, or about 99.9% sequence identity to SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17, or SEQ ID NO: 18. In someembodiments, nucleotide sequences for FusR1, homologs of FusR1,orthologs of FusR1, paralogs of FusR1, and fragments and variationsthereof have the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 17, or SEQ ID NO: 18.

In some embodiments, nucleotide sequences for FusR1, homologs of FusR1,orthologs of FusR1, paralogs of FusR1, and fragments and variationsthereof can be used to be expressed in plants. In some embodiments, saidnucleotide sequences can be used to be incorporated into an expressioncassette, which is capable of directing expression of a nucleotidesequence for FusR1, homologs of FusR1, orthologs of FusR1, paralogs ofFusR1, and fragments and variations thereof in a plant cell, forexample, banana varieties disclosed herein. This expression cassettecomprises a promoter operably linked to the nucleotide sequence ofinterest (i.e. FusR1, orthologs of FusR1, and fragments and variationsthereof) which is operably linked to termination signals. It alsotypically comprises sequences required for proper translation of thenucleotide sequence. The coding region usually codes for a protein ofinterest, (i.e. FUSR1). In some embodiments, the expression cassettecomprising the nucleotide sequence for FusR1, homologs of FusR1,orthologs of FusR1, paralogs of FusR1, and fragments and variationsthereof is chimeric so that at least one of its components isheterologous with respect to at least one of its other components.

In other embodiments, the expression cassette is one which is naturallyoccurring but has been obtained in a recombinant form useful forheterologous expression. The expression of the nucleotide sequence inthe expression cassette can be under the control of a constitutivepromoter or of an inducible promoter which initiates transcription onlywhen the host cell is exposed to some particular external stimulus.Also, the expression of the nucleotide sequence in the expressioncassette can be under the control of a tissue-specific promoter. In thecase of a multicellular organism, the promoter can also be specific to aparticular tissue or organ or stage of development in animal and/orplant including banana species.

The present disclosure provides polypeptides and amino acid sequencescomprising at least a portion of the proteins encoded by nucleotidesequences for FusR1, homologs of FusR1, orthologs of FusR1, paralogs ofFusR1, and fragments and variations thereof.

The present disclosure also provides an amino acid sequence encoded bythe nucleic acid sequences of FusR1, homologs of FusR1, orthologs ofFusR1, paralogs of FusR1, and/or fragments and variations thereof. Insome embodiments, the present disclosure provides an isolatedpolypeptide comprising an amino acid sequence that shares at least about70%, about 75%, about 80%, about 85%, at least about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%,about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9%identity to an amino acid sequence encoded by the nucleic acid sequencesof FusR1, homologs of FusR1, orthologs of FusR1, paralogs of FusR1,and/or fragments and variations thereof. In one embodiment, the presentdisclosure provides an isolated polypeptide comprising an amino acidsequence which encodes an amino acid sequence that shares at least about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%,about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9%identity to an amino acid sequence encoded by the nucleic acid sequencesof FusR1, homologs of FusR1, orthologs of FusR1, paralogs of FusR1,and/or fragments and variations thereof.

The disclosure also encompasses variants and fragments of proteins of anamino acid sequence encoded by the nucleic acid sequences of FusR1,homologs of FusR1, orthologs of FusR1 and/or paralogs of FusR1. Thevariants may contain alterations in the amino acid sequences of theconstituent proteins. The term “variant” with respect to a polypeptiderefers to an amino acid sequence that is altered by one or more aminoacids with respect to a reference sequence. The variant can have“conservative” changes, or “nonconservative” changes, e.g., analogousminor variations can also include amino acid deletions or insertions, orboth.

Functional fragments and variants of a polypeptide include thosefragments and variants that maintain one or more functions of the parentpolypeptide. It is recognized that the gene or cDNA encoding apolypeptide can be considerably mutated without materially altering oneor more of the polypeptide's functions. First, the genetic code iswell-known to be degenerate, and thus different codons encode the sameamino acids. Second, even where an amino acid substitution isintroduced, the mutation can be conservative and have no material impacton the essential function(s) of a protein. See, e.g., StryerBiochemistry 3rd Ed., 1988. Third, part of a polypeptide chain can bedeleted without impairing or eliminating all of its functions. Fourth,insertions or additions can be made in the polypeptide chain forexample, adding epitope tags, without impairing or eliminating itsfunctions (Ausubel et al. J. Immunol. 159(5): 2502-12, 1997). Othermodifications that can be made without materially impairing one or morefunctions of a polypeptide can include, for example, in vivo or in vitrochemical and biochemical modifications or the incorporation of unusualamino acids. Such modifications include, but are not limited to, forexample, acetylation, carboxylation, phosphorylation, glycosylation,ubiquination, labelling, e.g., with radionucleotides, and variousenzymatic modifications, as will be readily appreciated by those wellskilled in the art. A variety of methods for labelling polypeptides, andlabels useful for such purposes, are well known in the art, and includeradioactive isotopes such as 32P, ligands which bind to or are bound bylabelled specific binding partners (e.g., antibodies), fluorophores,chemiluminescent agents, enzymes, and anti-ligands. Functional fragmentsand variants can be of varying length. For example, some fragments haveat least 10, 25, 50, 75, 100, 200, or even more amino acid residues.These mutations can be natural or purposely changed. In someembodiments, mutations containing alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the proteins or how the proteins are made are anembodiment of the disclosure.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions.Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Furtherinformation about conservative substitutions can be found, for instance,in Ben Bassat et al. (J. Bacteriol., 169:751 757, 1987), O'Regan et al.(Gene, 77:237 251, 1989), Sahin Toth et al. (Protein Sci., 3:240 247,1994), Hochuli et al. (Bio/Technology, 6:1321 1325, 1988) and in widelyused textbooks of genetics and molecular biology. The Blosum matricesare commonly used for determining the relatedness of polypeptidesequences. The Blosum matrices were created using a large database oftrusted alignments (the BLOCKS database), in which pairwise sequencealignments related by less than some threshold percentage identity werecounted (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919,1992). A threshold of 90% identity was used for the highly conservedtarget frequencies of the BLOSUM90 matrix. A threshold of 65% identitywas used for the BLOSUM65 matrix. Scores of zero and above in the Blosummatrices are considered “conservative substitutions” at the percentageidentity selected. The following table 2 shows exemplary conservativeamino acid substitutions.

TABLE 2 Exemplary conservative amino acid substitutions listed VeryHighly - Highly Conserved Original Conserved Substitutions (from theConserved Substitutions Residue Substitutions Blosum90 Matrix) (from theBlosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg LysGln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Lys,Ser, Arg, Asp, Gln, Glu, His, Lys, Thr Ser, Thr Asp Glu Asn, Glu Asn,Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Lys, Arg,Asn, Asp, Glu, His, Lys, Met Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn,Asp, Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln,Tyr Arg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met, Phe,Val Leu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys Arg; Gln; GluArg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln, Ile, Leu,Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, Tyr Ile, Leu,Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu, Gly, Lys,Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, Tyr Phe, TyrTyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala,Ile, Leu, Met, Thr

In some examples, variants can have no more than 3, 5, 10, 15, 20, 25,30, 40, 50, or 100 conservative amino acid changes (such as very highlyconserved or highly conserved amino acid substitutions). In otherexamples, one or several hydrophobic residues (such as Leu, Ile, Val,Met, Phe, or Trp) in a variant sequence can be replaced with a differenthydrophobic residue (such as Leu, Ile, Val, Met, Phe, or Trp) to createa variant functionally similar to the disclosed an amino acid sequencesencoded by the nucleic acid sequences of FusR1, homologs of FusR1,orthologs of FusR1 and/or paralogs of FusR1, and/or fragments andvariations thereof.

In some embodiments, variants may differ from the disclosed sequences byalteration of the coding region to fit the codon usage bias of theparticular organism into which the molecule is to be introduced. Inother embodiments, the coding region may be altered by taking advantageof the degeneracy of the genetic code to alter the coding sequence suchthat, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequencesubstantially similar to the disclosed an amino acid sequences encodedby the nucleic acid sequences of FusR1, homologs of FusR1, orthologs ofFusR1 and/or paralogs of FusR1, and/or fragments and variations thereof.

In some embodiments, functional fragments derived from the FusR1orthologs of the present disclosure are provided. The functionalfragments can still confer resistance to pathogens when expressed in aplant. In some embodiments, the functional fragments contain at leastthe conserved region or Bowman-Birk inhibitor domain of a wild typeFusR1 orthologs, or functional variants thereof. In some embodiments,the functional fragments contain one or more conserved region shared bytwo or more FusR1 orthologs, shared by two or more FusR1 orthologs inthe same plant genus, shared by two or more dicot FUSR1 orthologs,and/or shared by two or more monocot FusR1 orthologs. The conservedregions or Bowman-Birk inhibitor domains can be determined by anysuitable computer program, such as NCBI protein BLAST program and NCBIAlignment program, or equivalent programs. In some embodiments, thefunctional fragments are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50or more amino acids shorter compared to the FusR1 orthologs of thepresent disclosure. In some embodiments, the functional fragments aremade by deleting one or more amino acid of the FusR1 orthologs of thepresent disclosure. In some embodiments, the functional fragments shareat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity to theFusR1 orthologs of the present disclosure.

In some embodiments, functional chimeric or synthetic polypeptidesderived from the FusR1 orthologs of the present disclosure are provided.The functional chimeric or synthetic polypeptides can still conferresistance to pathogens when expressed in a plant. In some embodiments,the functional chimeric or synthetic polypeptides contain at least theconserved region or Bowman-Birk inhibitor domain of a wild type FUSR1orthologs, or functional variants thereof. In some embodiments, thefunctional chimeric or synthetic polypeptides contain one or moreconserved region shared by two or more FUSR1 orthologs, shared by two ormore FusR1 orthologs in the same plant genus, shared by two or moremonocot FusR1 orthologs, and/or shared by two or more dicot FUSR1orthologs. Non-limiting exemplary conserved regions are shown in FIG. 2.The conserved regions or Bowman-Birk inhibitor domains can be determinedby any suitable computer program, such as NCBI protein BLAST program andNCBI Alignment program, or equivalent programs. In some embodiments, thefunctional chimeric or synthetic polypeptides share at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or more identity to the FusR1 orthologs ofthe present disclosure.

Sequences of conserved regions unique to FW-sensitive alleles can alsobe used to knock-down the level of one or more FusR1 orthologs. In someembodiments, sequences of conserved regions can be used to make genesilencing molecules to target one or more FusR1 orthologs. In someembodiments, the gene silencing molecules are selected from the groupconsisting of double-stranded polynucleotides, single-strandedpolynucleotides or Mixed Duplex Oligonucleotides. In some embodiments,the gene silencing molecules comprises a DNA/RNA fragment of about 10bp, 15 bp, 19 bp, 20 bp, 21 bp, 25 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70bp, 80 bp, 90 bp, 100 bp, 150 bp, 200 pb, 250 bp, 300 bp, 350 bp, 400bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, or morepolynucleotides, wherein the DNA/RNA fragment share at least 90%, 95%,99%, or more identity to a conserved region of the FusR1 orthologssequences of the present disclosure, or complementary sequences thereof.

V. Plant Transformation

The present polynucleotides coding for FUSR1, homologs of FusR1,orthologs of FusR1 and/or paralogs of FusR1, and/or fragments andvariations thereof of the present disclosure can be transformed intobanana or other plant genera.

Methods of producing transgenic plants are well known to those ofordinary skill in the art. Transgenic plants can now be produced by avariety of different transformation methods including, but not limitedto, electroporation; microinjection; microprojectile bombardment, alsoknown as particle acceleration or biolistic bombardment; viral-mediatedtransformation; and Agrobacterium-mediated transformation. See, forexample, U.S. Pat. Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880;5,550,318; 5,641,664; 5,736,369 and 5,736,369; International PatentApplication Publication Nos. WO2002/038779 and WO/2009/117555; Lu etal., (Plant Cell Reports, 2008, 27:273-278); Watson et al., RecombinantDNA, Scientific American Books (1992); Hinchee et al., Bio/Tech.6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama etal., Bio/Tech. 6:1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839(1990); Mullins et al., Bio/Tech. 8:833-839 (1990); Hiei et al., PlantMolecular Biology 35:205-218 (1997); Ishida et al., Nature Biotechnology14:745-750 (1996); Zhang et al., Molecular Biotechnology 8:223-231(1997); Ku et al., Nature Biotechnology 17:76-80 (1999); and, Raineri etal., Bio/Tech. 8:33-38 (1990)), each of which is expressly incorporatedherein by reference in their entirety.

Agrobacterium tumefaciens is a naturally occurring bacterium that iscapable of inserting its DNA (genetic information) into plants,resulting in a type of injury to the plant known as crown gall. Mostspecies of plants can now be transformed using this method, includingcucurbitaceous species.

Microprojectile bombardment is also known as particle acceleration,biolistic bombardment, and the gene gun (Biolistic® Gene Gun). The genegun is used to shoot pellets that are coated with genes (e.g., fordesired traits) into plant seeds or plant tissues in order to get theplant cells to then express the new genes. The gene gun uses an actualexplosive (.22 caliber blank) to propel the material. Compressed air orsteam may also be used as the propellant. The Biolistic® Gene Gun wasinvented in 1983-1984 at Cornell University by John Sanford, EdwardWolf, and Nelson Allen. It and its registered trademark are now owned byE. I. du Pont de Nemours and Company. Most species of plants have beentransformed using this method.

The most common method for the introduction of new genetic material intoa plant genome involves the use of living cells of the bacterialpathogen Agrobacterium tumefaciens to literally inject a piece of DNA,called transfer or T-DNA, into individual plant cells (usually followingwounding of the tissue) where it is targeted to the plant nucleus forchromosomal integration. There are numerous patents governingAgrobacterium mediated transformation and particular DNA deliveryplasmids designed specifically for use with Agrobacterium—for example,U.S. Pat. No. 4,536,475, EP0265556, EP0270822, WO8504899, WO8603516,U.S. Pat. No. 5,591,616, EP0604662, EP0672752, WO8603776, WO9209696,WO9419930, WO9967357, U.S. Pat. No. 4,399,216, WO8303259, U.S. Pat. No.5,731,179, EP068730, WO9516031, U.S. Pat. Nos. 5,693,512, 6,051,757 andEP904362A1. Agrobacterium-mediated plant transformation involves as afirst step the placement of DNA fragments cloned on plasmids into livingAgrobacterium cells, which are then subsequently used for transformationinto individual plant cells. Agrobacterium-mediated plant transformationis thus an indirect plant transformation method. Methods ofAgrobacterium-mediated plant transformation that involve using vectorswith no T-DNA are also well known to those skilled in the art and canhave applicability in the present disclosure. See, for example, U.S.Pat. No. 7,250,554, which utilizes P-DNA instead of T-DNA in thetransformation vector.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome, although multiplecopies are possible. Such transgenic plants can be referred to as beinghemizygous for the added gene. A more accurate name for such a plant isan independent segregant, because each transformed plant represents aunique T-DNA integration event (U.S. Pat. No. 6,156,953). A transgenelocus is generally characterized by the presence and/or absence of thetransgene. A heterozygous genotype in which one allele corresponds tothe absence of the transgene is also designated hemizygous (U.S. Pat.No. 6,008,437).

Direct plant transformation methods using DNA have also been reported.The first of these to be reported historically is electroporation, whichutilizes an electrical current applied to a solution containing plantcells (M. E. Fromm et al., Nature, 319, 791 (1986); H. Jones et al.,Plant Mol. Biol., 13, 501 (1989) and H. Yang et al., Plant Cell Reports,7, 421 (1988). Another direct method, called “biolistic bombardment”,uses ultrafine particles, usually tungsten or gold, that are coated withDNA and then sprayed onto the surface of a plant tissue with sufficientforce to cause the particles to penetrate plant cells, including thethick cell wall, membrane and nuclear envelope, but without killing atleast some of them (U.S. Pat. Nos. 5,204,253, 5,015,580). A third directmethod uses fibrous forms of metal or ceramic consisting of sharp,porous or hollow needle-like projections that literally impale thecells, and also the nuclear envelope of cells. Both silicon carbide andaluminum borate whiskers have been used for plant transformation (Mizunoet al., 2004; Petolino et al., 2000; U.S. Pat. No. 5,302,523 USApplication 20040197909) and also for bacterial and animaltransformation (Kaepler et al., 1992; Raloff, 1990; Wang, 1995). Thereare other methods reported, and undoubtedly, additional methods will bedeveloped. However, the efficiencies of each of these indirect or directmethods in introducing foreign DNA into plant cells are invariablyextremely low, making it necessary to use some method for selection ofonly those cells that have been transformed, and further, allowinggrowth and regeneration into plants of only those cells that have beentransformed.

For efficient plant transformation, a selection method must be employedsuch that whole plants are regenerated from a single transformed celland every cell of the transformed plant carries the DNA of interest.These methods can employ positive selection, whereby a foreign gene issupplied to a plant cell that allows it to utilize a substrate presentin the medium that it otherwise could not use, such as mannose or xylose(for example, refer U.S. Pat. Nos. 5,767,378; 5,994,629). Moretypically, however, negative selection is used because it is moreefficient, utilizing selective agents such as herbicides or antibioticsthat either kill or inhibit the growth of non-transformed plant cellsand reducing the possibility of chimeras. Resistance genes that areeffective against negative selective agents are provided on theintroduced foreign DNA used for the plant transformation. For example,one of the most popular selective agents used is the antibiotickanamycin, together with the resistance gene neomycin phosphotransferase(nptll), which confers resistance to kanamycin and related antibiotics(see, for example, Messing & Vierra, Gene 19: 259-268 (1982); Bevan etal., Nature 304:184-187 (1983)). However, many different antibiotics andantibiotic resistance genes can be used for transformation purposes(refer U.S. Pat. Nos. 5,034,322, 6,174,724 and 6,255,560). In addition,several herbicides and herbicide resistance genes have been used fortransformation purposes, including the bar gene, which confersresistance to the herbicide phosphinothricin (White et al., Nucl AcidsRes 18: 1062 (1990), Spencer et al., Theor Appl Genet 79: 625-631(1990),U.S. Pat. Nos. 4,795,855, 5,378,824 and 6,107,549). In addition, thedhfr gene, which confers resistance to the anticancer agentmethotrexate, has been used for selection (Bourouis et al., EMBO J.2(7): 1099-1104 (1983).

The expression control elements used to regulate the expression of agiven protein can either be the expression control element that isnormally found associated with the coding sequence (homologousexpression element) or can be a heterologous expression control element.A variety of homologous and heterologous expression control elements areknown in the art and can readily be used to make expression units foruse in the present disclosure. Transcription initiation regions, forexample, can include any of the various opine initiation regions, suchas octopine, mannopine, nopaline and the like that are found in the Tiplasmids of Agrobacterium tumefaciens. Alternatively, plant viralpromoters can also be used, such as the cauliflower mosaic virus 19S and35S promoters (CaMV 19S and CaMV 35S promoters, respectively) to controlgene expression in a plant (U.S. Pat. Nos. 5,352,605; 5,530,196 and5,858,742 for example). Enhancer sequences derived from the CaMV canalso be utilized (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938;5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example). Lastly,plant promoters such as prolifera promoter, fruit specific promoters,Ap3 promoter, heat shock promoters, seed specific promoters, etc. canalso be used.

Either a gamete-specific promoter, a constitutive promoter (such as theCaMV or Nos promoter), an organ-specific promoter (such as the E8promoter from tomato), or an inducible promoter is typically ligated tothe protein or antisense encoding region using standard techniques knownin the art. The expression unit may be further optimized by employingsupplemental elements such as transcription terminators and/or enhancerelements.

Thus, for expression in plants, the expression units will typicallycontain, in addition to the protein sequence, a plant promoter region, atranscription initiation site and a transcription termination sequence.Unique restriction enzyme sites at the 5′ and 3′ ends of the expressionunit are typically included to allow for easy insertion into apre-existing vector.

In the construction of heterologous promoter/structural gene orantisense combinations, the promoter is preferably positioned about thesame distance from the heterologous transcription start site as it isfrom the transcription start site in its natural setting. As is known inthe art, however, some variation in this distance can be accommodatedwithout loss of promoter function.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes. If the mRNA encoded by the structural gene is tobe efficiently processed, DNA sequences which direct polyadenylation ofthe RNA are also commonly added to the vector construct. Polyadenylationsequences include, but are not limited to the Agrobacterium octopinesynthase signal (Gielen et al., EMBO J3:835-846 (1984)) or the nopalinesynthase signal (Depicker et al., Mol. and Appl. Genet. 1:561-573(1982)). The resulting expression unit is ligated into or otherwiseconstructed to be included in a vector that is appropriate for higherplant transformation. One or more expression units may be included inthe same vector. The vector will typically contain a selectable markergene expression unit by which transformed plant cells can be identifiedin culture. Usually, the marker gene will encode resistance to anantibiotic, such as G418, hygromycin, bleomycin, kanamycin, orgentamicin or to an herbicide, such as glyphosate (Round-Up) orglufosinate (BASTA) or atrazine. Replication sequences, of bacterial orviral origin, are generally also included to allow the vector to becloned in a bacterial or phage host; preferably a broad host range forprokaryotic origin of replication is included. A selectable marker forbacteria may also be included to allow selection of bacterial cellsbearing the desired construct. Suitable prokaryotic selectable markersinclude resistance to antibiotics such as ampicillin, kanamycin ortetracycline. Other DNA sequences encoding additional functions may alsobe present in the vector, as is known in the art. For instance, in thecase of Agrobacterium transformations, T-DNA sequences will also beincluded for subsequent transfer to plant chromosomes.

To introduce a desired gene or set of genes by conventional methodsrequires a sexual cross between two lines, and then repeatedback-crossing between hybrid offspring and one of the parents until aplant with the desired characteristics is obtained. This process,however, is restricted to plants that can sexually hybridize, and genesin addition to the desired gene will be transferred.

Recombinant DNA techniques allow plant researchers to circumvent theselimitations by enabling plant geneticists to identify and clone specificgenes for desirable traits, such as improved fatty acid composition, andto introduce these genes into already useful varieties of plants. Oncethe foreign genes have been introduced into a plant, that plant can thenbe used in imp plant breeding schemes (e.g., pedigree breeding,single-seed-descent breeding schemes, reciprocal recurrent selection) toproduce progeny which also contain the gene of interest.

Genes can be introduced in a site directed fashion using homologousrecombination. Homologous recombination permits site-specificmodifications in endogenous genes and thus inherited or acquiredmutations may be corrected, and/or novel alterations may be engineeredinto the genome. Homologous recombination and site-directed integrationin plants are discussed in, for example, U.S. Pat. Nos. 5,451,513;5,501,967 and 5,527,695.

According to Ploetz (2015, Phytopathology 105:1512-1521), “Genetictransformation of bananas has become commonplace, and disease resistanceis one of the most sought-after traits [citations omitted].” Techniquesfor transforming and regenerating banana plants are well known in theart. See, for example, U.S. Pat. Nos. 7,534,930; 6,133,035; Sagi et al.,Bio/Technology 13, 481-485, 1995; May et al., Bio/Technology 13,485-492, 1995; Vishnevetsky et al., Transgenic Res. 20(1):61-71, 2011;Paul et al. (2011); Zhong et al., Plant Physiol. 110, 1097-1107, 1996;and, Dugdale et al., Journal of General Virology 79:2301-2311, 1998,each of which is expressly incorporated herein by reference in theirentirety. For overviews and history, see, for example, Mohan and Swennen(editors), 2004, Banana improvement: cellular, molecular biology, andinduced mutations, Science Publishers, Inc.; and, Remy et al., 2013,Genetically modified bananas: Past, present and future, ActaHorticulturae 974:71-80, each of which is expressly incorporated hereinby reference in their entirety.

While reducing the present invention to practice, the inventor canconstruct an expression construct which includes nucleotide sequencesencoding FUSR1, homologs of FusR1, orthologs of FusR1 and/or paralogs ofFusR1, and/or fragments and variations thereof. The expression constructof the present invention can be introduced into embryogenic callus ofcommercial banana and the resulting transformed cells can be regeneratedinto plants. The transgenic banana plants is expected to have expressionof FW-resistant FUSR1 protein and pathogen resistance.

According to one aspect of the present invention, there is provided amethod of producing a disease resistant banana plant. The method iseffected by transforming a banana cell with at least one exogenouspolynucleotide encoding a polypeptide (such as FW-resistant FusR1)capable of conferring disease resistance to a banana plant.

According to another aspect of the present invention, there is provideda method of producing a disease resistant banana plant. The method iseffected by transforming a banana cell with at least one exogenousexpression cassette containing polynucleotides encoding aCRISPR-associated effector protein and a guide RNA capable of targetingat least one FW-sensitive FusR1 allele, thereby conferring diseaseresistance to a banana plant.

The banana cell of the present invention can be any banana variety orcultivar, including, but not limited to, commercially important M.acuminata (Cavendish, dwarf Cavendish, Grand Nain etc.). Preferably, thebanana cell used for transformation is an embryogenic cell which iscapable of forming a whole plant. More preferably, the banana cell is anembryogenic callus cell.

The phrase “embryogenic callus cell” used herein refers to anembryogenic cell contained in a cell mass produced in vitro.

Banana embryogenic callus cells suitable for transformation can begenerated using well known methodology. For example, immature maleflowers (inflorescences) can be dissected and incubated in M1 medium(see content in Table 1 herein below) under a reduced light intensity(50-100 lux) at 25° C. Following 3-5 months of incubation in M1 medium,yellow embryogenic calli are transferred to M2 medium (see content inTable 1 below) and incubated at 27° C. in the dark for at least fourmonths to promote embryogenesis.

As is mentioned hereinabove, such banana embryogenic callus cells aresuitable for transformation with a nucleic acid construct which includesat least one polynucleotide encoding a disease resistance polypeptide.

The phrases “polypeptide capable of conferring disease resistance” and“disease resistance polypeptide” are interchangeably used herein torefer to any peptide, polypeptide or protein which is capable ofprotecting a banana plant (expressing the polypeptide) from pathogeninfection or the harmful effects resultant from pathogen infection.

A suitable disease resistance polypeptide can also be a polypeptidecapable of inducing or enhancing resistance in plants such as described,for example in U.S. Pat. Nos. 6,091,004 and 6,316,697.

As is mentioned hereinabove, the method of the present invention iseffected by transforming a banana cell with at least one polynucleotideencoding a polypeptide capable of conferring disease resistance to abanana plant.

In some embodiments, the banana cell is transformed with apolynucleotide sequence encoding FUSR1 protein from Musa itinerans, anexample of which is set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:4, and SEQ ID NO: 5.

In some embodiments, the banana cell is transformed with apolynucleotide sequence encoding FUSR1 protein from Musa acuminata, anexample of which is set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10 and SEQ ID NO: 11

In some embodiments, the banana cell is transformed with apolynucleotide sequence encoding FUSR1 protein from Musa basjoo, anexample of which is set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 20, and SEQ ID NO: 21.

In some embodiments, the banana cell is transformed with apolynucleotide sequence encoding FUSR1 protein from Musella lasiocarpa,an example of which is set forth in SEQ ID NO: 23.

In some embodiments, the banana cell is transformed with apolynucleotide sequence encoding FUSR1 protein from Musa balbisiana, anexample of which is set forth in SEQ ID NO: 26.

In some embodiments, plants transformed with just a single exogenousdisease-resistance polypeptide, such as FUSR1, may exhibit only partialand short-lasting protection (see, for example, in Jach et al., Plant J.8:97-108, 1995). In other embodiments, the banana cell/plant of thepresent invention preferably expresses a plurality of exogenous diseaseresistance polypeptides and is thus substantially more disease resistantthan unmodified plants.

Several approaches can be utilized to transform and co-express thesepolynucleotides in plant cells.

Although less preferred, each of the above described polynucleotidesequences can be separately introduced into a banana cell by using threeseparate nucleic-acid constructs. In some embodiments, the threepolynucleotide sequences can be co-introduced and co-expressed in thebanana cell using a single nucleic acid construct. Such a construct canbe designed with a single promoter sequences co-which can transcribe apolycistronic message including all three polynucleotide sequences. Toenable co-translation of the three polypeptides encoded by thepolycistronic message, the polynucleotide sequences can be inter-linkedvia an internal ribosome entry site (IRES) sequence which facilitatestranslation of polynucleotide sequences positioned downstream of theIRES sequence. In this case, a transcribed polycistronic RNA moleculeencoding the three polypeptides described above will be translated fromboth the capped 5′ end and the two internal IRES sequences of thepolycistronic RNA molecule to thereby produce in the cell all threepolypeptides.

Alternatively, the polynucleotide segments encoding the plurality ofpolypeptides capable of conferring disease resistance can betranslationally fused via a protease recognition site cleavable by aprotease expressed by the cell to be transformed with the nucleic acidconstruct. In this case, a chimeric polypeptide translated will becleaved by a cell-expressed protease to thereby generate the pluralityof polypeptides.

In other embodiments, the present invention utilizes a nucleic acidconstruct which includes three promoter sequences each capable ofdirecting transcription of a specific polynucleotide sequence of thepolynucleotide sequences described above.

Suitable promoters which can be used with the nucleic acid of thepresent invention include constitutive, inducible, or tissue-specificpromoters.

Suitable constitutive promoters include, for example, CaMV 35S promoter(Odell et al., Nature 313:810-812, 1985); maize Ubi 1 (Christensen etal., Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al.,Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet.81:581-588, 1991); and Synthetic Super MAS (Ni et al., The Plant Journal7: 661-76, 1995). Other constitutive promoters include those in U.S.Pat. Nos. 5,659,026, 5,608,149; 5,608,144; 5,604,121; 5,569,597:5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Suitable inducible promoters can be pathogen-inducible promoters suchas, for example, the alfalfa PR10 promoter (Coutos-Thevenot et al.,Journal of Experimental Botany 52: 901-910, 2001 and the promotersdescribed by Marineau et al., Plant Mol. Biol. 9:335-342, 1987; Mattonet al. Molecular Plant-Microbe Interactions 2:325-331, 1989; Somsisch etal., Proc. Natl. Acad. Sci. USA 83:2427-2430, 1986: Somsisch et al.,Mol. Gen. Genet. 2:93-98, 1988; and Yang, Proc. Natl. Acad. Sci. USA93:14972-14977, 1996.

Suitable tissue-specific promoters include, but not limited to,leaf-specific promoters such as described, for example, by Yamamoto etal., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67,1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor etal., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol.23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA90:9586-9590, 1993.

The nucleic acid construct of the present invention may also include atleast one selectable marker such as, for example, nptII. Preferably, thenucleic acid construct is a shuttle vector, which can propagate both inE. coli (wherein the construct comprises an appropriate selectablemarker and origin of replication) and be compatible for propagation incells. The construct according to the present invention can be, forexample, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus oran artificial chromosome, preferably a plasmid.

The nucleic acid construct of the present invention can be utilized tostably transform banana cells. The principle methods of causing stableintegration of exogenous DNA into banana genome include two mainapproaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; glass fibers or silicon carbide whiskertransformation of cell cultures, embryos or callus tissue, U.S. Pat. No.5,464,765 or by the direct incubation of DNA with germinating pollen,DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman,G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. Horsch et al. in Plant Molecular Biology Manual A5,Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementaryapproach employs the Agrobacterium delivery system in combination withvacuum infiltration. Suitable Agrobacterium-mediated procedures forintroducing exogenous DNA to banana cells is described by Dougale et al.(Journal of General Virology, 79:2301-2311, 1998) and in U.S. Pat. No.6,395,962.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues.

Alternatively, the nucleic acid construct of the present invention canbe introduced into banana cells by a microprojectiles bombardment. Inthis technique, tungsten or gold particles coated with exogenous DNA areaccelerated toward the target cells. Suitable banana transformationprocedures by microprojectiles bombardment are described by Sagi et al.(Biotechnology 13:481-485, 1995) and by Dougale et al. (Journal ofGeneral Virology, 79:2301-2311, 1998). Preferably, the nucleic acidconstruct of the present invention is introduced into banana cells by amicroprojectiles bombardment procedure as described in Example 4 hereinbelow.

Following transformation, the transformed cells are micropropagated toprovide a rapid, consistent reproduction of the transformed material.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant. Micropropagationallows mass production of quality plant material in a short period oftime and offers a rapid multiplication of selected cultivars in thepreservation of the characteristics of the original transgenic ortransformed plant. The advantages of cloning plants are the speed ofplant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration ofculture medium or growth conditions between stages. Thus, themicropropagation process involves four basic stages: Stage one, initialtissue culturing; stage two, tissue culture multiplication; stage three,differentiation and plant formation; and stage four, greenhouseculturing and hardening. During stage one, initial tissue culturing, thetissue culture is established and certified contaminant-free. Duringstage two, the initial tissue culture is multiplied until a sufficientnumber of tissue samples are produced to meet production goals. Duringstage three, the tissue samples grown in stage two are divided and growninto individual plantlets. At stage four, the transformed plantlets aretransferred to a greenhouse for hardening where the plants' tolerance tolight is gradually increased so that it can be grown in the naturalenvironment.

Thus, transformed banana cells can be micropropagated and regeneratedinto plants using methods known in the art such as described, forexample in U.S. Pat. No. 6,133,035 and by Novak et al., 1989; Dhed'a etal., 1991; Cote et al., 1996; Becker et al., 2000; Sagi et al. PlantCell Reports 13:262-266, 1994; Grapin et al., Cell Dev. Biol. Plant.32:66-71, 1996; Marroquin et al., In Vivo Cell. Div. Biol. 29P:43-46,1993; and Escalant et al., In Vivo Cell Dev. Biol. 30:181-186, 1994).

Stable integration of exogenous DNA sequence in the genome of thetransformed plants can be determined using standard molecular biologytechniques well known in the art such as PCR and Southern blothybridization.

Although stable transformation is presently preferred, transienttransformation of cultured cells, leaf cells, meristematic cells or thewhole plant is also envisaged by the present invention.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viral infection is preferred since is enables circumventingmicropropagation and regeneration of a whole plant from cultured cells.Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman et al. (Communications in MolecularBiology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp.172-189, 1988). Pseudovirus particles for use in expressing foreign DNAin many hosts, including plants, is described in WO 87/06261.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous nucleic acid sequences in plants is demonstrated bythe above references as well as by Dawson et al. (Virology 172:285-292,1989; Takamatsu et al. EMBO J. 6:307-311, 1987; French et al. (Science231:1294-1297, 1986); and Takamatsu et al. (FEBS Letters 269:73-76,1990).

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA.

If the virus is an RNA virus, the virus is generally cloned as a cDNAand inserted into a plasmid. The plasmid is then used to make all of theconstructions. The RNA virus is then produced by transcribing the viralsequence of the plasmid and translation of the viral genes to producethe coat protein(s) which encapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous nucleic acid sequences such as thoseincluded in the construct of the present invention is demonstrated bythe above references as well as in U.S. Pat. No. 5,316,931.

In one embodiment, a plant viral nucleic acid is provided in which thenative coat protein coding sequence has been deleted from a viralnucleic acid, a non-native plant viral coat protein coding sequence anda non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral nucleic acid, andensuring a systemic infection of the host by the recombinant plant viralnucleic acid, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native nucleic acid sequencewithin it, such that a protein is produced. The recombinant plant viralnucleic acid may contain one or more additional non-native subgenomicpromoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or nucleic acid sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) nucleic acid sequencesmay be inserted adjacent the native plant viral subgenomic promoter orthe native and a non-native plant viral subgenomic promoters if morethan one nucleic acid sequence is included. The non-native nucleic acidsequences are transcribed or expressed in the host plant under controlof the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral nucleic acid isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that the sequences are transcribed or expressed in the host plantunder control of the subgenomic promoters to produce the desiredproduct.

In a fourth embodiment, a recombinant plant viral nucleic acid isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral nucleic acid to produce a recombinant plantvirus. The recombinant plant viral nucleic acid or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral nucleic acid is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(isolated nucleic acid) in the host to produce the desired protein.

In addition to the above, the nucleic acid molecule of the presentinvention can also be introduced into a chloroplast genome therebyenabling chloroplast expression.

A technique for introducing exogenous nucleic acid sequences to thegenome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theexogenous nucleic acid is introduced via particle bombardment into thecells with the aim of introducing at least one exogenous nucleic acidmolecule into the chloroplasts. The exogenous nucleic acid is selectedsuch that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the exogenous nucleic acid includes, inaddition to a gene of interest, at least one nucleic acid stretch whichis derived from the chloroplast's genome. In addition, the exogenousnucleic acid includes a selectable marker, which serves by sequentialselection procedures to ascertain that all or substantially all of thecopies of the chloroplast genomes following such selection will includethe exogenous nucleic acid. Further details relating to this techniqueare found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which areincorporated herein by reference. A polypeptide can thus be produced bythe protein expression system of the chloroplast and become integratedinto the chloroplast's inner membrane.

In case that the exogenous polypeptide confers disease resistance to theplant, the expression can be determined based on increased in resistanceor tolerance to pathogens, preferably in comparison with similarwild-type (non-transformed) plant. Comparative evaluation of plants fortheir resistance or tolerance to pathogens can be effected using invitro or in vivo bioassays well known in the art of plant pathology suchas described, for example by Agrios, G. N., ed. (Plant Pathology, ThirdEdition, Academic Press, New York, 1988).

Evaluating plant resistance or tolerance to pathogens can be effected byexposing a pathogen to an extract obtained from plant tissue anddetermining the effect of the extract on the pathogen growth in vitro.In some embodiments, evaluating plant resistance or tolerance topathogens is effected by exposing a pathogen to a plant tissue (e.g., aleaf tissue).

In other embodiments, evaluating plant resistance or tolerance topathogens is effected by exposing a pathogen to a whole plant. Forexample, evaluating plant resistance or tolerance to Fusarium oxysporumf. sp. Cubense (Foc) (the causal agent of Panama disease) can beeffected by planting transformed banana plants in an open field in aclose proximity to non-transformed plants which are infected with thepathogen (used as a source of inoculum). The disease severity whichsubsequently develops in transformed plants is evaluated comparativelyto non-transformed plants. The disease severity is preferably evaluatedvisually (the damage usually appears on suckers which have at least 5-12leaves) and statistically analyzed to determine significant differencesin resistance or tolerance between plant lines to the Panama disease.

Hence, the present invention provides nucleic acid constructs includingone or more polynucleotides encoding disease resistance polypeptides,transformed banana cells and transformed banana plants expressingexogenous disease resistance traits, and methods of producing same.

VI. Breeding Methods

Open-Pollinated Populations. The improvement of open-pollinatedpopulations of such crops as rye, many maizes and sugar beets, herbagegrasses, legumes such as alfalfa and clover, and tropical tree cropssuch as cacao, coconuts, oil palm and some rubber, depends essentiallyupon changing gene-frequencies towards fixation of favorable alleleswhile maintaining a high (but far from maximal) degree ofheterozygosity. Uniformity in such populations is impossible andtrueness-to-type in an open-pollinated variety is a statistical featureof the population as a whole, not a characteristic of individual plants.Thus, the heterogeneity of open-pollinated populations contrasts withthe homogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes for flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement. First, there is the situation in which a population ischanged en masse by a chosen selection procedure. The outcome is animproved population that is indefinitely propagable by random-matingwithin itself in isolation. Second, the synthetic variety attains thesame end result as population improvement but is not itself propagableas such; it has to be reconstructed from parental lines or clones. Theseplant breeding procedures for improving open-pollinated populations arewell known to those skilled in the art and comprehensive reviews ofbreeding procedures routinely used for improving cross-pollinated plantsare provided in numerous texts and articles, including: Allard,Principles of Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds,Principles of Crop Improvement, Longman Group Limited (1979); Hallauerand Miranda, Quantitative Genetics in Maize Breeding, Iowa StateUniversity Press (1981); and, Jensen, Plant Breeding Methodology, JohnWiley & Sons, Inc. (1988). For population improvement methods specificfor soybean see, e.g., J. R. Wilcox, editor (1987) SOYBEANS:Improvement, Production, and Uses, Second Edition, American Society ofAgronomy, Inc., Crop Science Society of America, Inc., and Soil ScienceSociety of America, Inc., publishers, 888 pages.

Mass Selection. In mass selection, desirable individual plants arechosen, harvested, and the seed composited without progeny testing toproduce the following generation. Since selection is based on thematernal parent only, and there is no control over pollination, massselection amounts to a form of random mating with selection. As statedabove, the purpose of mass selection is to increase the proportion ofsuperior genotypes in the population.

Synthetics. A synthetic variety is produced by crossing inter se anumber of genotypes selected for good combining ability in all possiblehybrid combinations, with subsequent maintenance of the variety by openpollination. Whether parents are (more or less inbred) seed-propagatedlines, as in some sugar beet and beans (Vicia) or clones, as in herbagegrasses, clovers and alfalfa, makes no difference in principle. Parentsare selected on general combining ability, sometimes by test crosses ortoperosses, more generally by polycrosses. Parental seed lines may bedeliberately inbred (e.g. by selfing or sib crossing). However, even ifthe parents are not deliberately inbred, selection within lines duringline maintenance will ensure that some inbreeding occurs. Clonal parentswill, of course, remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or two cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

Hybrids. As discussed above, hybrid is an individual plant resultingfrom a cross between parents of differing genotypes. Commercial hybridsare now used extensively in many crops, including corn (maize), sorghum,sugar beet, sunflower and broccoli. Hybrids can be formed in a number ofdifferent ways, including by crossing two parents directly (single crosshybrids), by crossing a single cross hybrid with another parent(three-way or triple cross hybrids), or by crossing two differenthybrids (four-way or double cross hybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

Bulk Segregation Analysis (BSA). BSA, a.k.a. bulked segregationanalysis, or bulk segregant analysis, is a method described byMichelmore et al. (Michelmore et al., 1991, Identification of markerslinked to disease-resistance genes by bulked segregant analysis: a rapidmethod to detect markers in specific genomic regions by usingsegregating populations. Proceedings of the National Academy ofSciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie et al., Bulksegregant analysis with molecular markers and its use for improvingdrought resistance in maize, 1999, Journal of Experimental Botany,50(337):1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to pathogen), and theother from the individuals having reversed phenotype (e.g., susceptibleto pathogen), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs). Markers linked to thephenotype can be identified and used for breeding or QTL mapping.

Gene Pyramiding. The method to combine into a single genotype a seriesof target genes identified in different parents is usually referred asgene pyramiding. The first part of a gene pyramiding breeding is calleda pedigree and is aimed at cumulating one copy of all target genes in asingle genotype (called root genotype). The second part is called thefixation steps and is aimed at fixing the target genes into a homozygousstate, that is, to derive the ideal genotype (ideotype) from the rootgenotype. Gene pyramiding can be combined with marker assisted selection(MAS, see Hospital et al., 1992, 1997a, and 1997b, and Moreau et al,1998) or marker based recurrent selection (MBRS, see Hospital et al.,2000).

Banana breeding programs, especially for edible bananas, is hampered byhigh sterility, triploidy and seedlessness. Few diploid banana clonesproduce viable pollen, and the germplasm of commercial banana clones isboth male- and female-sterile. In spite of these problems andchallenges, important progress has been made in the genetic improvementof Musa in recent years, and new varieties are not becoming availablefrom banana breeding programs (Escalant and Jain, Chapter 30, Bananaimprovement with cellular and molecular biology, and induced mutations:future and perspectives, 8 pages, In Jain and Swennan, editors, BananaImprovement: Cellular, Molecular Biology, and Induced Mutations, 2004,Food and Agriculture Organization of the United Nations, SciencePublishers, Inc.).

For information on banana breeding see, for example, Heslop-Harrison andSchwarzacher, Annals of Botany 100:1073-1084, 2007; Bakry et al.,Chapter 1, Genetic Improvement in Banana, 50 pages, In BreedingPlantation Tree Crops: Tropical Species, 2009; Heslop-Harrison et al.,Genomics, Banana Breeding and Superdomestication, Acta Hort. 897:55-62,2011; Jenny et al., In Jacome et al., editors, Mycosphaerella leaf spotdiseases of banana: present status and outlook, Proceedings of the2^(nd) International Workshop on Mycosphaerella leaf spot diseases heldin San Jose, Costa Rica, 20-23 May 2002, Session 4, pages 199-208; Ortizet al., Banana and Plantain Breeding, Chapter 10, pages 110-146, InGowen et al., editors, Bananas and Plantains, World Crop Series,Springer Link, 1995; Batte et al., Frontiers in Plant Science, Volum 10,Article 81, 9 pages, February 2019.

VII. Gene Editing

As used herein, the term “gene editing system” refers to a systemcomprising one or more DNA-binding domains or components and one or moreDNA-modifying domains or components, or isolated nucleic acids, e.g.,one or more vectors, encoding said DNA-binding and DNA-modifying domainsor components. Gene editing systems are used for modifying the nucleicacid of a target gene and/or for modulating the expression of a targetgene. In known gene editing systems, for example, the one or moreDNA-binding domains or components are associated with the one or moreDNA-modifying domains or components, such that the one or moreDNA-binding domains target the one or more DNA-modifying domains orcomponents to a specific nucleic acid site. Methods and compositions forenhancing gene editing is well known in the art. See example, U.S.Patent Application Publication No. 2018/0245065, which is incorporatedby reference in its entirety.

Certain gene editing systems are known in the art, and include but arenot limited to, zinc finger nucleases, transcription activator-likeeffector nucleases (TALEN5); clustered regularly interspaced shortpalindromic repeats (CRISPR)/Cas systems, meganuclease systems, andviral vector-mediated gene editing.

In some embodiments, the present disclosure teaches methods for geneediting/cloning utilizing DNA nucleases. CRISPR complexes, transcriptionactivator-like effector nucleases (TALEN5), zinc finger nucleases(ZFNs), and FokI restriction enzymes, which are some of thesequence-specific nucleases that have been used as gene editing tools.These enzymes are able to target their nuclease activities to desiredtarget loci through interactions with guide regions engineered torecognize sequences of interest. In some embodiments, the presentdisclosure teaches CRISPR-based gene editing methods to geneticallyengineer the genome of banana species of the present disclosure in orderto stimulate, enhance, or modulate disease resistance to pathogens.

(i) CRISPR Systems

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) andCRISPR-associated (cas) endonucleases were originally discovered asadaptive immunity systems evolved by bacteria and archaea to protectagainst viral and plasmid invasion. Naturally occurring CRISPR/Cassystems in bacteria are composed of one or more Cas genes and one ormore CRISPR arrays consisting of short palindromic repeats of basesequences separated by genome-targeting sequences acquired frompreviously encountered viruses and plasmids (called spacers).(Wiedenheft, B., et. al. Nature. 2012; 482:331; Bhaya, D., et. al.,Annu. Rev. Genet. 2011; 45:231; and Terms, M. P. et. al., Curr. Opin.Microbiol. 2011; 14:321). Bacteria and archaea possessing one or moreCRISPR loci respond to viral or plasmid challenge by integrating shortfragments of foreign sequence (protospacers) into the host chromosome atthe proximal end of the CRISPR array. Transcription of CRISPR locigenerates a library of CRISPR-derived RNAs (crRNAs) containing sequencescomplementary to previously encountered invading nucleic acids(Haurwitz, R. E., et. al., Science. 2012:329; 1355; Gesner, E. M., et.al., Nat. Struct. Mol. Biol. 2001:18; 688; Jinek, M., et. al., Science.2012:337; 816-21). Target recognition by crRNAs occurs throughcomplementary base pairing with target DNA, which directs cleavage offoreign sequences by means of Cas proteins. (Jinek et. al. 2012 “AProgrammable dual-RNA-guided DNA endonuclease in adaptive bacterialimmunity.” Science. 2012:337; 816-821).

There are at least five main CRISPR system types (Type I, II, III, IVand V) and at least 16 distinct subtypes (Makarova, K. S., et al., NatRev Microbiol. 2015. Nat. Rev. Microbiol. 13, 722-736). CRISPR systemsare also classified based on their effector proteins. Class 1 systemspossess multi-subunit crRNA-effector complexes, whereas in Class 2systems all functions of the effector complex are carried out by asingle protein (e.g., Cas9 or Cpf1). In some embodiments, the presentdisclosure provides using type II and/or type V single-subunit effectorsystems.

As these naturally occur in many different types of bacteria, the exactarrangements of the CRISPR and structure, function and number of Casgenes and their product differ somewhat from species to species. Haft etal. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol.8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al.(2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151:653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. Forexample, the Cse (Cas subtype, E. coli) proteins (e.g., CasA) form afunctional complex, Cascade, which processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. Brouns et al. (2008) Science321: 960-964. In other prokaryotes, Cas6 processes the CRISPRtranscript. The CRISPR-based phage inactivation in E. coli requiresCascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module)proteins in Pyrococcus furiosus and other prokaryotes form a functionalcomplex with small CRISPR RNAs that recognizes and cleaves complementarytarget RNAs. A simpler CRISPR system relies on the protein Cas9, whichis a nuclease with two active cutting sites, one for each strand of thedouble helix. Combining Cas9 and modified CRISPR locus RNA can be usedin a system for gene editing. Pennisi (2013) Science 341: 833-836.

(ii) CRISPR/Cas9

In some embodiments, the present disclosure provides methods of geneediting using a Type II CRISPR system. Type II systems rely on a i)single endonuclease protein, ii) a transactiving crRNA (tracrRNA), andiii) a crRNA where a ˜20-nucleotide (nt) portion of the 5′ end of crRNAis complementary to a target nucleic acid. The region of a CRISPR crRNAstrand that is complementary to its target DNA protospacer is herebyreferred to as “guide sequence.”

In some embodiments, the tracrRNA and crRNA components of a Type IIsystem can be replaced by a single guide RNA (sgRNA), also known as aguide RNA (gRNA). The sgRNA can include, for example, a nucleotidesequence that comprises an at least 12-20 nucleotide sequencecomplementary to the target DNA sequence (guide sequence) and caninclude a common scaffold RNA sequence at its 3′ end. As used herein, “acommon scaffold RNA” refers to any RNA sequence that mimics the tracrRNAsequence or any RNA sequences that function as a tracrRNA.

Cas9 endonucleases produce blunt end DNA breaks, and are recruited totarget DNA by a combination of a crRNA and a tracrRNA oligos, whichtether the endonuclease via complementary hybridization of the RNACRISPR complex.

In some embodiments, DNA recognition by the crRNA/endonuclease complexrequires additional complementary base-pairing with a protospaceradjacent motif (PAM) (e.g., 5′-NGG-3′) located in a 3′ portion of thetarget DNA, downstream from the target protospacer. (Jinek, M., et. al.,Science. 2012, 337:816-821). In some embodiments, the PAM motifrecognized by a Cas9 varies for different Cas9 proteins.

In some embodiments the Cas9 disclosed herein can be any variant derivedor isolated from any source. In other embodiments, the Cas9 peptide ofthe present disclosure can include one or more of the mutationsdescribed in the literature, including but not limited to the functionalmutations described in: Fonfara et al. Nucleic Acids Res. 2014 February;42(4):2577-90; Nishimasu H. et al. Cell. 2014 Feb. 27, 156(5):935-49;Jinek M. et al. Science. 2012 337:816-21; and Jinek M. et al. Science.2014 Mar. 14, 343(6176); see also U.S. patent application Ser. No.13/842,859, filed Mar. 15, 2013, which is hereby incorporated byreference; further, see U.S. Pat. Nos. 8,697,359; 8,771,945; 8,795,965;8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814;8,945,839; 8,993,233; and 8,999,641, which are all hereby incorporatedby reference. Thus, in some embodiments, the systems and methodsdisclosed herein can be used with the wild type Cas9 protein havingdouble-stranded nuclease activity, Cas9 mutants that act as singlestranded nickases, or other mutants with modified nuclease activity.

According to the present disclosure, Cas9 molecules of, derived from, orbased on the Cas9 proteins of a variety of species can be used in themethods and compositions described herein. For example, Cas9 moleculesof, derived from, or based on, e.g., S. pyogenes, S. thermophilus,Staphylococcus aureus and/or Neisseria meningitidis Cas9 molecules, canbe used in the systems, methods and compositions described herein.Additional Cas9 species include: Acidovorax avenae, Actinobacilluspleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis,Actinomyces sp., cychphilus denitrificans, Aminomonas paucivorans,Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroidessp., Blastopirellula marina, Bradyrhiz obium sp., Brevibacilluslatemsporus, Campylobacter coli, Campylobacter jejuni, Campylobacterlad, Candidatus Puniceispirillum, Clostridiu cellulolyticum, Clostridiumperfringens, Corynebacterium accolens, Corynebacterium diphtheria,Corynebacterium matruchotii, Dinoroseobacter sliibae, Eubacteriumdolichum, gamma proteobacterium, Gluconacetobacler diazotrophicus,Haemophilus parainfluenzae, Haemophilus sputorum, Helicobactercanadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobaclerpolytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii,Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp.,Methylosinus trichosporium, Mobiluncus mulieris, Neisseriabacilliformis, Neisseria cinerea, Neisseria flavescens, Neisserialactamica. Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp.,Parvibaculum lavamentivorans, Pasteurella multocida,Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonaspalustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp.,Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcussp., Subdoligranulum sp., Tislrella mobilis, Treponema sp., orVerminephrobacter eiseniae.

In some embodiments, the present disclosure teaches the use of tools forgenome editing techniques in plants such as crops and methods of geneediting using CRISPR-associated (cas) endonucleases including SpyCas9,SaCas9, St1Cas9. These powerful tools for genome editing, which can beapplied to plant genome editing are well known in the art. See example,Song et al. (2016), CRISPR/Cas9: A powerful tool for crop genomeediting, The Crop Journal 4:75-82, Mali et al. (2013) RNA-guided humangenome engineering via cas9, Science 339: 823-826; Ran et al. (2015) Invivo genome editing using Staphylococcus aureus cas9, Nature 520:186-191; Esvelt et al. (2013) Orthogonal cas9 proteins for ma-guidedgene regulation and editing, Nature methods 10(11): 1116-1121, each ofwhich is hereby incorporated by reference in its entirety for allpurposes.

(iii) CRISPR/Cpf1

In other embodiments, the present disclosure provides methods of geneediting using a Type V CRISPR system. In some embodiments, the presentdisclosure provides methods of gene editing using CRISPR fromPrevotella, Francisella, Acidaminococcus, Lachnospiraceae, and Moraxella(Cpf1).

The Cpf1 CRISPR systems of the present disclosure comprise i) a singleendonuclease protein, and ii) a crRNA, wherein a portion of the 3′ endof crRNA contains the guide sequence complementary to a target nucleicacid. In this system, the Cpf1 nuclease is directly recruited to thetarget DNA by the crRNA. In some embodiments, guide sequences for Cpf1must be at least 12nt, 13nt, 14nt, 15nt, or 16nt in order to achievedetectable DNA cleavage, and a minimum of 14nt, 15nt, 16nt, 17nt, or18nt to achieve efficient DNA cleavage.

The Cpf1 systems of the present disclosure differ from Cas9 in a varietyof ways. First, unlike Cas9, Cpf1 does not require a separate tracrRNAfor cleavage. In some embodiments, Cpf1 crRNAs can be as short as about42-44 bases long—of which 23-25 nt is guide sequence and 19 nt is theconstitutive direct repeat sequence. In contrast, the combined Cas9tracrRNA and crRNA synthetic sequences can be about 100 bases long.

Second, certain Cpf1 systems prefer a “TTN” PAM motif that is located 5′upstream of its target. This is in contrast to the “NGG” PAM motifslocated on the 3′ of the target DNA for common Cas9 systems such asStreptococcus pyogenes Cas9. In some embodiments, the uracil baseimmediately preceding the guide sequence cannot be substituted (Zetsche,B. et al. 2015. “Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2CRISPR-Cas System” Cell 163, 759-771, which is hereby incorporated byreference in its entirety for all purposes).

Third, the cut sites for Cpf1 are staggered by about 3-5 bases, whichcreate “sticky ends” (Kim et al., 2016. “Genome-wide analysis revealsspecificities of Cpf1 endonucleases in human cells” published onlineJun. 6, 2016). These sticky ends with 3-5 nt overhangs are thought tofacilitate NHEJ-mediated-ligation, and improve gene editing of DNAfragments with matching ends. The cut sites are in the 3′ end of thetarget DNA, distal to the 5′ end where the PAM is. The cut positionsusually follow the 18th base on the non-hybridized strand and thecorresponding 23rd base on the complementary strand hybridized to thecrRNA.

Fourth, in Cpf1 complexes, the “seed” region is located within the first5 nt of the guide sequence. Cpf1 crRNA seed regions are highly sensitiveto mutations, and even single base substitutions in this region candrastically reduce cleavage activity (see Zetsche B. et al. 2015 “Cpf1Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System” Cell163, 759-771). Critically, unlike the Cas9 CRISPR target, the cleavagesites and the seed region of Cpf1 systems do not overlap. Additionalguidance on designing Cpf1 crRNA targeting oligos is available onZetsche B. et al. 2015. (“Cpf1 Is a Single RNA-Guided Endonuclease of aClass 2 CRISPR-Cas System” Cell 163, 759-771).

(iv) Guide RNA (gRNA)

In some embodiments, the guide RNA of the present disclosure comprisestwo coding regions, encoding for crRNA and tracrRNA, respectively. Inother embodiments, the guide RNA is a single guide RNA (sgRNA) syntheticcrRNA/tracrRNA hybrid. In other embodiments, the guide RNA is a crRNAfor a Cpf1 endonuclease.

Persons having skill in the art will appreciate that, unless otherwisenoted, all references to a single guide RNA (sgRNA) in the presentdisclosure can be read as referring to a guide RNA (gRNA). Therefore,embodiments described in the present disclosure which refer to a singleguide RNA (sgRNA) will also be understood to refer to a guide RNA(gRNA).

The guide RNA is designed so as to recruit the CRISPR endonuclease to atarget DNA region. In some embodiments, the present disclosure teachesmethods of identifying viable target CRISPR landing sites, and designingguide RNAs for targeting the sites. For example, in some embodiments,the present disclosure teaches algorithms designed to facilitate theidentification of CRISPR landing sites within target DNA regions.

In some embodiments, the present disclosure teaches use of softwareprograms designed to identify candidate CRISPR target sequences on bothstrands of an input DNA sequence based on desired guide sequence lengthand a CRISPR motif sequence (PAM, protospacer adjacent motif) for aspecified CRISPR enzyme. For example, target sites for Cpf1 fromFrancisella novicida U112, with PAM sequences TTN, may be identified bysearching for 5′-TTN-3′ both on the input sequence and on thereverse-complement of the input. The target sites for Cpf1 fromLachnospiraceae bacterium and Acidaminococcus sp., with PAM sequencesTTTN, may be identified by searching for 5′-TTTN-3′ both on the inputsequence and on the reverse complement of the input. Likewise, targetsites for Cas9 of S. thermophilus CRISPR, with PAM sequence NNAGAAW, maybe identified by searching for 5′-Nx-NNAGAAW-3′ both on the inputsequence and on the reverse-complement of the input. The PAM sequencefor Cas9 of S. pyogenes is 5′-NGG-3′.

Since multiple occurrences in the genome of the DNA target site may leadto nonspecific genome editing, after identifying all potential sites,sequences may be filtered out based on the number of times they appearin the relevant reference genome or modular CRISPR construct. For thoseCRISPR enzymes for which sequence specificity is determined by a ‘seed’sequence (such as the first 5 bp of the guide sequence for Cpf1-mediatedcleavage) the filtering step may also account for any seed sequencelimitations.

In some embodiments, algorithmic tools can also identify potential offtarget sites for a particular guide sequence. For example, in someembodiments Cas-Offinder can be used to identify potential off targetsites for Cpf1 (see Kim et al., 2016. “Genome-wide analysis revealsspecificities of Cpf1 endonucleases in human cells” Nature Biotechnology34, 863-868). Any other publicly available CRISPR design/identificationtool may also be used, including for example the Zhang labcrispr.mit.edu tool (see Hsu, et al. 2013 “DNA targeting specificity ofRNA guided Cas9 nucleases” Nature Biotech 31, 827-832).

In some embodiments, the user may be allowed to choose the length of theseed sequence. The user may also be allowed to specify the number ofoccurrences of the seed: PAM sequence in a genome for purposes ofpassing the filter. The default is to screen for unique sequences.Filtration level is altered by changing both the length of the seedsequence and the number of occurrences of the sequence in the genome.The program may in addition or alternatively provide the sequence of aguide sequence complementary to the reported target sequence(s) byproviding the reverse complement of the identified target sequence(s).

In the guide RNA, the “spacer/guide sequence” sequence is complementaryto the “proto spacer” sequence in the DNA target. The gRNA” scaffold”for a single stranded gRNA structure is recognized by the Cas9 protein.

In some embodiments, the transgenic plant, plant part, plant cell, orplant tissue culture taught in the present disclosure comprise arecombinant construct, which comprises at least one nucleic acidsequence encoding a guide RNA. In some embodiments, the nucleic acid isoperably linked to a promoter. In other embodiments, a recombinantconstruct further comprises a nucleic acid sequence encoding a Clusteredregularly interspaced short palindromic repeats (CRISPR) endonuclease.In other embodiments, the guide RNA is capable of forming a complex withsaid CRISPR endonuclease, and said complex is capable of binding to andcreating a double strand break in a genomic target sequence of saidplant genome. In other embodiments, the CRISPR endonuclease is Cas9.

In further embodiments, the target sequence is a nucleic acid for FusR1,homologs of FusR1, orthologs of FusR1 and/or paralogs of FusR1, and/orfragments and variations thereof. In some embodiments, the presentdisclosure teaches the gene editing of FusR1 in FW-sensitive bananavarieties susceptible to Fusarium pathogens using genetic engineeringtechniques described herein.

The present disclosure teaches the targeted gene-editing techniques formodulating, stimulating, and enhancing disease resistance by turningFW-sensitive alleles to FW-resistant alleles based on sequenceinformation given in the present disclosure. The present disclosureteaches sequence information of both FW-resistant alleles andFW-sensitive alleles. Using CRISPR/Cas system, FW-resistant traits areintroduced into FW-sensitive banana varieties.

In some embodiments, FW-sensitive FusR1 alleles are to be targeted forknock-out. In some embodiments, sequences of conserved regionsresponsible for FW sensitivity trait can be used to make gene editingmachineries (such as CRISPR-associated effector proteins, ZFN, TALENetc.) to target one or more FusR1 orthologs.

In some embodiments, the disrupting of expression of the endogenousFW-sensitive alleles is carried out by a gene-editing technology. Insome embodiments, the knock-out of FW-sensitive alleles is carried outby gene-editing technology. In some embodiments, the base-editing ofFW-sensitive alleles into FW-resistant alleles is carried out bygene-editing technology. In some embodiments, the gene-editingtechnology is a ZFN. In other embodiments, the gene-editing technologyis a TALEN. In further embodiments, the gene-editing technology is aCRISPR/Cas system. In further embodiments, said CRISPR system comprisesa nucleic acid molecule and an enzymatic protein, wherein the nucleicacid molecule is a guide RNA (gRNA) molecule and the enzymatic proteinis a Cas protein or Cas ortholog. In further embodiments, at least twoexpression cassettes are stacked in tandem in the expression vector.

In some embodiments, the modified plant cells comprise one or moremodifications (e.g., insertions, deletions, or mutations of one or morenucleic acids) in the genomic DNA sequence of an endogenous target generesulting in the altered function the endogenous gene, therebymodulating, stimulating, or enhancing disease resistance. In suchembodiments, the modified plant cells comprise a “modified endogenoustarget gene.” In some embodiments, the modifications in the genomic DNAsequence cause mutation, thereby altering the function of FW-sensitiveFUSR1 protein to FW-resistant FUSR1 protein. In some embodiments, themodifications in the genomic DNA sequence results in amino acidsubstitutions, thereby altering the normal function of the encodedprotein. In some embodiments, the modifications in the genomic DNAsequence encode a modified endogenous protein with modulated, altered,stimulated or enhanced function of disease/pathogen resistance comparedto the unmodified (i.e., FW-sensitive) version of the endogenous proteinin the FW-sensitive banana accessions.

In some embodiments, the modified plant cells described herein compriseone or more modified endogenous target genes, wherein the one or moremodifications result in an altered function of a gene product (i.e., aprotein) encoded by the endogenous target gene compared to an unmodifiedplant cell. For example, in some embodiments, a modified plant celldemonstrates expression of a FW-resistant FUSR1 protein or anupregulated expression of said protein. In some embodiments, theexpression of the gene product (such as genetically-engineeredFW-resistant FusR1 from FW-sensitive FusR1) in a modified plant cell isenhanced by at least 0.5%, 1%, 2%, 3%, 4%, 5% or higher compared to theexpression of the gene product (such as FW-sensitive FusR1) in anunmodified plant cell. In other embodiments, the expression of the geneproduct (such as genetically-engineered FW-resistant FusR1) in amodified plant cell is enhanced by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more compared to the expression of the geneproduct (such as FW-sensitive FusR1) in an unmodified plant cell. Insome embodiments, the modified plant cells described herein demonstrateenhanced expression and/or function of gene products encoded by aplurality (e.g., two or more) of endogenous target genes compared to theexpression of the gene products in an unmodified plant cell. Forexample, in some embodiments, a modified plant cell demonstratesenhanced expression and/or function of gene products from 2, 3, 4, 5, 6,7, 8, 9, 10, or more endogenous target genes compared to the expressionof the gene products in an unmodified plant cell.

In some embodiments, the modified plant cells described herein compriseone or more modified endogenous target genes, wherein the one or moremodifications to the target DNA sequence results in expression of aprotein with reduced or altered function (e.g., a “modified endogenousprotein”) compared to the function of the corresponding proteinexpressed in an unmodified plant cell (e.g., a “unmodified endogenousprotein”). In some embodiments, the modified plant cells describedherein comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified endogenoustarget genes encoding 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modifiedendogenous proteins. In some embodiments, the modified endogenousprotein demonstrates enhanced or altered binding affinity for anotherprotein expressed by the modified plant cell or expressed by anothercell; enhanced or altered signaling capacity; enhanced or alteredenzymatic activity; enhanced or altered DNA-binding activity; or reducedor altered ability to function as a scaffolding protein.

EXAMPLES

The present invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety for all purposes.

Example 1: Methods and Materials for Sequencing

(1) Material

Fresh and lyophilized banana leaf tissues were obtained from BioversityInternational (Leuven, Belgium), Inter-TROP CRB Plantes Tropicales(Guadeloupe), and the IITA Genebank (Ibadan, Nigeria), Plant DelightsNursery (Raleigh, N.C.), and The Flower Bin (Longmont, Colo.).

(2) RNA

Total RNA was extracted from fresh, frozen, and lyophilized bananaleaves using a modified Ishihara protocol (Ishihara et al., 2016).Approximately 100 mg of fresh or frozen banana tissue was ground to apowder using a clean, dry-ice cooled mortar and pestle that was treatedwith RNase Away™ (Invitrogen, Carlsbad, Calif.). Approximately 20-30 mgof lyophilized banana tissue was homogenized in a Lysing Matrix D Tube(MP Bio, Santa Ana, Calif.) without liquid. One milliliter of polyphenollysis buffer (800 μl RLT buffer (Qiagen, Germantown, Md.), 200 μl ofFruit-mate (Takara, Mountain View, Calif.), and 10 μl ofβ-mercaptoethanol) was added to each sample. Fresh and frozen sampleswere homogenized for 40 seconds on the speed 6 setting of a FastPrep 120(ThermoFisher Scientific, Waltham, Mass.), while lyophilized sampleswere vortexed on high for 1 minute. All samples were incubated on icefor 4 minutes, then centrifuged for 2 minutes at 8000×g. The supernatantwas transferred to a new 2.0 ml tube and another 1.0 ml of polyphenollysis buffer was added to the supernatant. Samples were vortexed on highfor 1 minute, incubated on ice for 4 minutes, and centrifuged for 2minutes at 8000 x g. The supernatant was split between two QIAshreddercolumns (Qiagen, Germantown, Md.) and centrifuged on maximum speed for 2minutes until all supernatant had been processed. The remaining steps ofRNA extraction were carried out according to the Ishihara protocol. Theoptional in-solution DNase digestion and RNA cleanup protocol was alsoperformed as detailed in the RNeasy Mini protocol (Qiagen, Germantown,Md.). Sample concentration and purity was determined using the NanoDrop™One (ThermoFisher Scientific, Waltham, Mass.) spectrophotometer.

(3) DNA

Total DNA was extracted from fresh, frozen, and lyophilized bananaleaves using a modified PowerPlant Pro DNA Isolation Kit protocol (MOBIO, Carlsbad, Calif.). Approximately 40 mg of fresh or frozen bananatissue was ground to a powder using a cleaned, dry-ice cooled mortar andpestle that was treated with RNase Away™ (Invitrogen, Carlsbad, Calif.).Approximately 10-20 mg of lyophilized banana tissue was homogenized in aLysing Matrix D Tube (MP Bio, Santa Ana, Calif.) without liquid. Theremaining steps of DNA extraction were carried out according to the MOBIO protocol. Phenolic Separation Solution was added to the lysis bufferand 250 μl of PD3 buffer was used. Sample concentration and purity wasdetermined using the NanoDrop™ One (ThermoFisher Scientific, Waltham,Mass.) spectrophotometer.

(4) cDNA

cDNA was synthesized from 1.0 μg of total RNA using the 1st Strand cDNASynthesis Kit (Epicentre, Madison, Wis.). The adapter primer (AP) fromInvitrogen's 3′-RACE kit (Invitrogen, Carlsbad, Calif.) was used inplace of the poly dT primer.

(5) Primers

Primer sequences were designed against homologous regions of putativetarget genes with annealing temperatures of 57°−64° C. using theOligoAnalyzer Tool (IDT, Coralville, Iowa) program. Primers werepurchased from IDT.

(6) PCR

PCR reactions were performed in 25 μl reactions containing a finalconcentration of 1× Phusion® HF buffer, 300 μM each dNTP, 0.3 μM eachforward and reverse primer, 0.5 Units 1× Phusion® High-Fidelity DNAPolymerase (ThermoFisher Scientific, Waltham, Mass.) in a Veriti ThermalCycler (Applied Biosystems, Carlsbad, Calif.). General PCR conditionswere 98° C. for 2 minutes, followed by 35 cycles of 98° C. for 10seconds, 55°−62° C. for 30 seconds (depending on primer Ta), and 72° C.for 30 seconds, before a final extension at 72° C. for 10 minutes and ahold at 4° C. PCR products were run on a 1.5% agarose gel and visualizedusing GelRed® Nucleic Acid Stain (Biotium, Hayward, Calif.) on an AlphaImager EC (Alpha Innotech, San Leandro, Calif.).

(7) Cloning

PCR fragments were cloned using the Zero Blunt TOPO PCR Cloning Kit(Invitrogen, Carlsbad, Calif.) using 4 μl of PCR product, according tothe manufacturer's protocol. The ligated vector was transformed intoTop10 One Shot chemically competent cells (Invitrogen, Carlsbad, Calif.)using the chemical transformation protocol. The transformed E. colicells were plated onto LB agar plates containing 50 μg/ml kanamycin andthe plates were cultured overnight at 37° C.

(8) Colony PCR

Colonies containing recombinant plasmids were screened using PCR withM13 forward and reverse primers. PCR reactions were performed in 15 μlvolumes containing 60 mM Tris-SO4 (pH 8.9), 18 mM Ammonium Sulfate, 2.0mM Magnesium Sulfate, 0.2 mM each dNTP, 0.2 μM each forward and reverseprimer, 0.3 Units Platinum Taq Hi Fidelity (Invitrogen, Carlsbad,Calif.) in a Veriti Thermal Cycler (Applied Biosystems, Carlsbad,Calif.). Colonies were picked and inoculated into the PCR reaction,followed by an inoculation of 50 μl of LB-kanamycin. The colony PCRconditions were 94° C. for 2 minutes, followed by 35 cycles of 94° C.for 30 seconds, 50° C. for 30 seconds, and 68° C. for 1 minute, before afinal extension at 68° C. for 10 minutes and a hold at 4° C. PCRproducts were run on a 1.5% agarose gel and visualized using GelRed®Nucleic Acid Stain (Biotium, Hayward, Calif.) on an Alpha Imager EC(Alpha Innotech, San Leandro, Calif.). Colony PCR reactions producingproducts of the expected size were sequenced.

(9) Sequencing

Five microliters of each PCR product was prepared for sequencing byenzymatic treatment using 2 μl of High-Throughput ExoSAP-IT (Affymetrix,Santa Clara, Calif.). Reactions were incubated at 37° C. for 15 minutes,followed by 15 minutes at 80° C. Template was labeled for sequencingusing the BigDye Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, Carlsbad, Calif.) as follows: 2 μl of the template and 2 μlof a 0.8 μM sequencing primer was added to a mixture of BigDyeTerminator sequencing buffer, BigDye Terminator v3.1 Ready Reaction Mix,and water, in a 10 μl reaction. The BigDye sequencing reactionconditions were as follows: 96° C. for 1 minute, followed by 25 cyclesof 96° C. for 10 seconds, 50° C. for 5 seconds, and 60° C. for 75seconds. Unincorporated BigDye terminators were removed using the BigDyeXTerminator Purification Kit (Applied Biosystems, Carlsbad, Calif.). Thereactions were sequenced using the Applied Biosystems 3500 GeneticAnalyzer (Applied Biosystems, Carlsbad, Calif.).

(10) Sequence Alignment

Sequence files from the ABI 3500 Genetic Analyzer were imported intoSequencher v4.8 Build 3767 (Gene Codes, Ann Arbor, Mich.). Vectorsequence was trimmed using the Trim Vector tool. Sequences were thenautomatically aligned and manually edited for sequencing artifacts.

Example 2: Identifying Structural Differences Between Fusarium Wilt(FW)-Resistant Gene(s) and Fusarium Wilt (FW)-Sensitive Gene(s)

In this example, Fusarium Wilt resistance genes were discovered byanalysis, as described below, of DNA sequences retrieved from GenBank.Nucleotide sequences from several banana species (i.e. Musa itinerans,Musa acuminata, Musa basjoo, Musella lasiocarpa, Musa balbisiana) weredownloaded. The M. itinerans FusR1 sequence was obtained from multipleaccessions (ITC1526, ITC1571, and PT-BA-00223), all of which areFW-resistant. The M. acuminata FusR1 sequence labeled 1‘FW-resistant’was obtained from multiple FW-resistant accessions, including ITC0896 (Ma. subspecies banksii) and PT_BA-00281 (Pisang Bangkahulu). The M.acuminata sequence labeled ‘sensitive’ is from the FW-sensitiveaccessions (ITC0507, ITC0685, PT-BA-00304, PT-BA-00310, andPT-BA-00315). These accessions include multiple samples from bananacultivars such as Pisang Madu, Pisang Pipit, and Pisang Rojo Uter, allof which have been well-characterized as FW-sensitive (Chen et al,2019). The M. balbisiana sequence was obtained from several FW-sensitiveaccessions, including ITC1016, ITC0545, ITC0080, and ITC0565. FusR1 fromM. basjoo is from FW-resistant accessions (ITC0061 and PD #3064).Automated bioinformatics analysis was then applied to each pairwisecomparison and only those sequences that contain a nucleotide change (orchanges) that yield evolutionarily significant change(s) were retainedfor further analysis. This enabled the identification of genes that haveevolved to confer some evolutionary advantage as well as theidentification of the specific evolved changes.

Any of several different molecular evolution analyses or Ka/Ks-typemethods can be employed to evaluate quantitatively and qualitatively theevolutionary significance of the identified nucleotide changes betweenhomologous gene sequences from related species (Kreitman and Akashi,1995; Li, 1997). For example, positive selection on proteins (i.e.,molecular-level adaptive evolution) can be detected in protein-codinggenes by pairwise comparisons of the ratios of nonsynonymous nucleotidesubstitutions per nonsynonymous site (Ka) to synonymous substitutionsper synonymous site (Ks) (Li et al., 1985; Li, 1993). Any comparison ofKa and Ks may be used, although it is particularly convenient and mosteffective to compare these two variables as a ratio. Sequences areidentified by exhibiting a statistically significant difference betweenKa and Ks using standard statistical methods.

In some aspects, the Ka/Ks analysis by Li et al. (1993) is used to carryout the present disclosure, although other analysis programs that candetect positively selected genes between species can also be used (Li etal. 1985; Li, 1993; Messier and Stewart, 1997; Nei, 1987).

The Ka/Ks method, which comprises a comparison of the rate ofnon-synonymous substitutions per non-synonymous site with the rate ofsynonymous substitutions per synonymous site between homologousprotein-coding regions of genes in terms of a ratio, is used to identifysequence substitutions that may be driven by adaptive selection asopposed to neutral substitutions during evolution. A synonymous(‘silent’) substitution is one that, owing to the degeneracy of thegenetic code, makes no change to the amino acid sequence encoded; anon-synonymous substitution results in an amino acid replacement. Theextent of each type of change can be estimated as Ka and Ks,respectively, the numbers of synonymous substitutions per synonymoussite and non-synonymous substitutions per non-synonymous site.Calculations of Ka/Ks may be performed manually or by using software. Anexample of suitable programs are Li93 (Li, 1993), or MEGA X: MolecularEvolutionary Genetics Analysis Across Computing Platforms (Kumar et al.,2018)

For the purpose of estimating Ka and Ks, either complete or partialprotein-coding sequences are used to calculate total numbers ofsynonymous and non-synonymous substitutions, as well as non-synonymousand synonymous sites. The length of the polynucleotide sequence analyzedcan be any appropriate length. Preferably, the entire coding sequence iscompared in order to determine any and all significant changes. Publiclyavailable computer programs, such as Li93 (Li, 1993), or MEGA X:Molecular Evolutionary Genetics Analysis Across Computing Platforms(Kumar et al., 2018) can be used to calculate the Ka and Ks values forall pairwise comparisons.

This analysis can be further adapted to examine sequences in a “slidingwindow’ fashion such that small numbers of important changes are notmasked by the whole sequence. “Sliding window’ refers to examination ofconsecutive, over lapping subsections of the gene (the subsections canbe of any length).

The comparison of non-synonymous and synonymous substitution rates iscommonly represented by the Ka/Ks ratio. Ka/Ks has been shown to be areflection of the degree to which adaptive evolution has been at work inthe sequence under study. Full length or partial segments of a codingsequence can be used for the Ka/Ks analysis. The higher the Ka/Ks ratio,the more likely that a sequence has undergone adaptive evolution and thenon-synonymous substitutions are evolutionarily significant. See, forexample, Messier and Stewart (1997).

Ka/Ks ratios significantly greater than one (1.0) strongly suggest thatpositive selection has fixed greater numbers of amino acid replacementsthan can be expected as a result of chance alone and is in contrast tothe most commonly observed pattern in which the ratio is less than orequal to one (Nei, 1987; Hughes and Nei, 1988; Messier and Stewart,1994; Kreitman and Akashi, 1995; Messier and Stewart, 1997). Ratios lessthan one generally signify the role of negative, or purifying selectionindicating that there is strong pressure on the primary structure offunctional, effective proteins to remain unchanged.

All methods for calculating Ka/Ks ratios are based on a pairwisecomparison of the number of nonsynonymous substitutions pernonsynonymous site to the number of synonymous substitutions persynonymous site for the protein-coding regions of homologous genes fromrelated species. Each method implements different corrections forestimating “multiple hits” (i.e., more than one nucleotide substitutionat the same site). Each method also uses different models for how DNAsequences change over evolutionary time. Thus, preferably, a combinationof results from different algorithms is used to increase the level ofsensitivity for detection of positively-selected genes and confidence inthe result.

It is understood that the methods described herein could lead to theidentification of banana polynucleotide sequences that are functionallyrelated to banana protein coding sequences. Such sequences may include,but are not limited to, non-coding sequences or coding sequences that donot encode proteins. These related sequences can be, for example,physically adjacent to the banana protein-coding sequences in the bananagenome, such as introns or 5′- and 3′-flanking sequences (includingcontrol elements such as promoters and enhancers). These relatedsequences may be obtained via searching a public genome database such asGenBank or, alternatively, by screening and sequencing an appropriategenomic library with a protein-coding sequence as a probe.

After candidate genes were identified, the nucleotide sequences of thegenes in each orthologous gene pair were carefully verified by standardDNA sequencing techniques and then Ka/Ks analysis was repeated for eachcarefully sequenced candidate gene pair. More specifically, the softwareran through all possible pairwise comparisons between putative orthologsof every gene from cultivated banana, Musa acuminata (AAA subgr.Cavendish) compared to the orthologs from the wild species, looking forhigh Ka/Ks ratios. The software BLASTed (in automated fashion) everymRNA sequence from cultivated banana against every sequence in thetranscriptome that was sequenced from a wild relative, for example, M.balbisiana. The software then performed Ka/Ks analysis for each genepair (i.e., each set of orthologs), flagging the gene pairs with highKa/Ks scores.

The software then compared every cultivated banana sequence againstevery sequence of another wild relative, for example, M. basjoo, againby doing a series of BLASTs and then sifting through for high Ka/Ksscores. It thus does this for the transcriptome sequence of all the wildspecies in succession. This gives a set of candidates (see below) forsubsequent analysis. The software next compared every gene sequence inthe transcriptome of M. balbisiana against every sequence of M. basjoo,again by doing a series of BLASTs, and then sifting through for highKa/Ks scores. It thus ultimately compared all of the expressed genesrepresented in the utilized cDNA libraries of every banana speciesagainst all the genes of every other banana species, both wild andcultivated, with the goal of finding every gene that shows evidence ofpositive selection.

The flagged gene pairs that emerged were then individually and carefullyre-sequenced in the lab to check the accuracy of the originalhigh-throughput reads to eliminate false positives.

Next, every remaining candidate gene pair with a high Ka/Ks score wasexamined to determine if the comparison was truly orthologous or just anartifactual false positive caused by a paralogous comparison.

Using the methodology described above, banana gene sequences availablein GenBank were analyzed to identify a positively-selected gene that hasnot been linked to FW-resistance trait in banana species in the art.Inventor identified and selected this gene to be expected to give riseto FW-resistance and then named it as Fusarium Resistance 1 (FusR1).Remarkably, inventor found an unusually high Ka/Ks ratio of 3.6 betweenthe FusR1 ortholog from the highly resistant wild banana relative M.itinerans and FusR1 from FW-sensitive Cavendish (M. acuminata).

Inventor obtained accessions of a number of types of bananas, includingboth banana cultivars and landraces, as well as wild (undomesticated)banana species from the genera Musa, Musella, and Ensete. These threegenera comprise the banana family Musaceae. Inventor made substantialefforts to obtain multiple samples of both Musa acuminata (“A”-genome)and M balbisiana (“B”-genome) accessions, in order to adequately sampleboth the taxonomic and geographic diversity of bananas. Inventorobtained accessions of most of the acuminata subspecies. In addition,for outgroup analysis, inventor obtained plant accessions from plantfamilies known to be closely related to Musaceae.

It is well recognized that some B-group banana species/varieties arehighly susceptible to Foc-TR4 (Chen et al., 2019), even while sometimesdisplaying desirable agronomic traits such as drought tolerance. Thebananas of the A-genome display a range of Fusarium-resistance,tolerance, and sensitivity, depending upon the particular species orcultivated variety. As a consequence, many wild banana species andcultivated banana varieties have been carefully and rigorouslycharacterized for resistance, tolerance, or sensitivity to TR4 (Li etal., 2012, Ssali et al., 2013, Li et al., 2015, Wu et al., 2016, Ribeiroet al., 2018, Niu et al., 2018, and Zuo et al. 2018).

Whenever possible inventor chose to prepare both RNA (for conversion tocDNA) and genomic DNA (gDNA). Most accessions were obtained as eitherfresh, frozen, or lyophilized samples, and this usually permittedsuccessful RNA extraction. For some samples, particularly when older orpartially degraded, only gDNA could be isolated. mRNA sequences and/orcoding sequence only), intron sequence, and some sequences (see SequenceListing) for a number of Musa, Musella, Ensete, and outgroup species areprovided herewith as described in Table 1 and in the sequence listings.Detailed descriptions of the methods are given in Methods and Materialssection of Example 1.

Cultivated bananas are the product of hybridization events betweenB-genome bananas (the Musa balbisiana group) and A-genome bananas (theM. acuminata group). It is well recognized that some B-group bananaspecies/varieties are susceptible to Foc-TR4 (Chen et al., 2019), evenwhile sometimes possessing desirable agronomic traits such as droughttolerance (REF). In contrast, the bananas of the A-genome display arange of Fusarium-resistance, tolerance, and sensitivity, depending uponthe particular species or cultivated variety. Some A-genome groupspecies, such as Musa itinerans and M. basjoo, have been shown to beextremely resistant to Foc-TR4 (Li et al., 2015; Wu et al., 2016), whilesome A-genome cultivars like Cavendish are exquisitely sensitive toFusarium.

Analysis of these sequences revealed an important result; which is thatall “A”-genome banana species (or cultivated banana varieties) that havebeen characterized as Fusarium-resistant share FusR1 sequences that fallinto a common group, while Fusarium-sensitive banana species/varietiesfall into a different group. Strikingly, every B-genome accessioninventor examined is ‘FW-sensitive’, and all the FusR1 sequences fromB-genome accessions are broken and/or damaged in some fashion with somecombination of coding-sequence base pair deletions. Often the deletionis either long in size such as 82 or 85 bp, however inventor also founda consistent single base deletion. These deletions alter the inferredprotein sequence by destroying reading frame, usually resulting in atruncated protein. In addition, all B-genome FusR1 coding sequencescontain an unspliced 84 bp intron, often appearing together with the85-bp deletion.

As to A-genome bananas, inventor found that A-genome accessions that areknown to be Foc-TR4 resistant all share a common FusR1 sequence group,while Foc-TR4-sensitive A-genome accessions all share a different FusR1sequence group.

This is strong evidence that FusR1 is responsible for the observeddisease-resistance patterns between Fusarium-resistant vs.Fusarium-sensitive species. The analyses in this example suggest thatdiffering resistance from sensitivity to Fusarium race 4 is stronglylinked with FusR1 sequence differences.

Further support for this comes from our examination of the few bananaspecies that have been characterized as ‘Fusarium Wilt-tolerant’. Thesespecies all have FusR1 sequences that fall into a third sequence group,all are intermediate between the Fusarium-resistant andFusarium-sensitive sequence groups.

The banana industry was forced in the 1950s to convert from its primarycultivar, Gros Michel, to the Cavendish cultivar when Fusarium (PanamaDisease) race 1 posed a critical threat to Gros Michel. Cavendish, whichis a half-sib to Gros Michel (both are “A” genome species), was found tobe resistant to race 1. Thus, the closely related Cavendish and GrosMichel cultivars show differing profiles of resistance to the variousFusarium races. (Both are sensitive to Foc-TR4, the current threat tothe banana industry.)

Inventor sequenced FusR1 from a number of Musa acuminata accessions. Ineach case, inventor cloned, as described in Example 1, the FusR1 geneand then sequenced multiple clones of the FusR1 gene. Some of these M.acuminata accessions have been well-characterized for Fusarium Wiltresistance/sensitivity. Inventor found three alleles for M. acuminataFusR1. The critical observation is that all Fusarium Wilt-resistantaccessions share similar FusR1 sequences. The two FusR1 alleles fromFW-resistant M. acuminata accessions are the Fusarium Wilt-resistantFusR1 allele or simply, the “Resistant Alleles” (SEQ ID NO: 8 and SEQ IDNO: 10). In contrast, all FW-sensitive M. acuminata accessions share adifferent allele, named the Fusarium Wilt Sensitive FusR1 Allele (SEQ IDNO: 13). The FW-resistant FusR1 alleles differ in just a few criticalnucleotide substitutions from the FW-sensitive allele. (See FIG. 1).This strongly suggests that Fusarium Wilt resistance/sensitivity iscontrolled by the particular FusR1 allele that a banana plant carries.

Example 3: Resistance Breeding of Banana

Tetraploid versions of FW-sensitive Cavendish cultivars (M. acuminata;AAAA) are available or can be developed via large pollination/breedingprograms focused on creating, identifying and isolating the relativelylow percentage of tetraploid progeny that are produced (e.g., AguilarMoran, J. F., 2013, Improvement of Cavendish Banana cultivars throughconventional breeding, Acta Hortic. 986:205-208; Jenny et al., In Jacomeet al., editors, Mycosphaerella leaf spot diseases of banana: presentstatus and outlook, Proceedings of the 2^(nd) International Workshop onMycosphaerella leaf spot diseases held in San Jose, Costa Rica, 20-23May 2002, Session 4, pages 199-208) or by subjecting diploid AAgenotypes to in vitro polyploidization (Amah et al., November 2019,Frontiers in Plant Science, Vol. 10, Article 1450, 12 pages).

Diploid versions of FW-resistant FusR1 (AA) of M. acuminata ssp. banksiacan be identified or developed using methods known to those skilled inthe art (e.g., Bakry et al., Chapter 1, Genetic Improvement in Banana,50 pages, In Breeding Plantation Tree Crops: Tropical Species, 2009).The resultant diploids are screened for the presence of SEQ ID NO: 8and/or SEQ ID NO: 10 (mRNA sequences).

A tetraploid FW-sensitive Cavendish plant, such as a tetraploid of the‘Naine’ or ‘Williams’ cultivar, can be used a male parent in crosseswith a diploid FW-resistant FusR1 M acuminata ssp. banksia plant, suchas a diploid ‘ITC0896,’ used as the female parent.

A large number of the resultant progeny are screened for triploid plants(AAA) comprising SEQ ID NO: 8 and/or SEQ ID NO: 10 (mRNA sequences) andsubsequently evaluated for agronomic traits.

All resulting/selected banana plants with resistance to TR4 can bemaintained via asexual reproduction and used for production or insubsequent breeding programs.

Example 4: Materials and Methods for Plant Transformation

Banana transformation systems will use sterile material of selectedbanana strains. A variety of tissue culture and transformationmethodologies will be used to increase the likelihood of success. See,for example, the transformation protocols described in Ploetz (2015,Phytopathology 105:1512-1521), U.S. Pat. Nos. 7,534,930; 6,133,035; Sagiet al., Bio/Technology 13, 481-485, 1995; May et al., Bio/Technology 13,485-492, 1995; Vishnevetsky et al., Transgenic Res. 20(1):61-71, 2011;Paul et al. (2011); Zhong et al., Plant Physiol. 110, 1097-1107, 1996;Dugdale et al., Journal of General Virology 79:2301-2311, 1998; Mohanand Swennen (editors), 2004, Banana improvement: cellular, molecularbiology, and induced mutations, Science Publishers, Inc.; and, Remy etal., 2013, Genetically modified bananas: Past, present and future, ActaHorticulturae 974:71-80, each of which is expressly incorporated hereinby reference in their entireties.

These methodologies will focus on tissue culture conditions, identifyingdifferent tissue types for regeneration/shooting, media formulations,agrobacterium strains, selection cassettes, constructing control anddelivery vectors, gene delivery, selectable markers, and targettissue/cell substrates for DNA delivery and transformation. Initialexperiments will deploy control vectors using visual markers andselection cassettes to rapidly optimize experimental direction andscreen potential transgenic events. Parallel experiments will bedirected at optimizing transformation efficiency and using genes ofinterest (GOI).

Modifications to media formulations, vectors, and transformationprocesses will be done to improve process and transformation efficiency.Transformation vectors that contain key genes of interest will continueto be transformed to produce additional overexpression or knock-outevents. Vectors to be used as necessary include but are not limited tomulti-gene stacked vectors, polycistronic gene vectors, and multi gRNACRISPR editing vectors for testing efficacy in banana. Testing will bedone on TO events to show presence and copy number of the selectablemarker gene or the GOI. In addition, mRNA expression analysis will beused as needed for any key GOIs. Putative transformed plant materialwill be used for subsequent testing or analysis.

CRISPR technologies are described in detail elsewhere herein, includingreferences to the compositions and procedures for using CRISPR to editplant genomes, such as the banana genome. Detailed compositions andprocedures for utilizing CRISPR to knock-out a gene in plants that givesrise to a phenotype of interest (e.g., resistance to fungal pathogenssuch as Fusarium) are provided in WO 2019/118342 (PCT/US2018/064735), WO2018/220581 (PCT/M2018/053903) and US 2019/0032070 (U.S. Ser. No.16/072,706), each of which is specifically and entirely incorporated byreference herein.

Once target sites for knocking out a candidate gene (e.g. endogenousFW-sensitive FusR1 gene(s)) are screened in silico and selected,CRISPR/Cas9 vectors for the targeted mutation(s) in the candidate genefound in plants of interest will be constructed for the transformationof the vectors into the plant of interest (i.e. FW-sensitive bananavarieties, such as the widely-grown triploid, sterile Cavendish varietyand its progeny).

The CRISPR/Cas9 vectors will be transformed into plants of interest suchas banana varieties, especially FW-sensitive bananas usingagrobacterium-mediated protocols that are known in the art (see forexample, Ma et al., 2015) and/or developed or refined by inventor.Tissue culture and regeneration of transformed plants will be performedaccordingly.

The transformed plants with the CRISPR/Cas9 vectors will be regeneratedand tested to verify the introduction of CRISPR/Cas9 vectors into theplant cells of interest. As a control for the induction of indels, aconstruct expressing wild-type Cas9 will also be used in thisexperiment.

The knock-out of the candidate gene(s) will be examined in alltransformed plants. The knock-out will be studied by (1) quantitativePCR to check suppression and/or silencing of the candidate gene or (2)PCR amplification and subsequent Sanger sequencing and/orhigh-throughput deep sequencing. Also, the amino acid substitution(s)caused by the introduced frame-shift to the target genome region will beanalyzed by protein sequencing with mass spectrometry.

The transformed plants obtained will be grown in the controlled greenhouse and/or field conditions. The transformed plants, verified withamino acid insertion, deletion, or substitution of interest, will beobserved for enhanced resistance to FW, Panama Disease, or infection byFusarium oxysporum f sp. cubense Tropical Race 4.

Example 5: Banana Transformation

Banana plants susceptible to Fusarium oxysporum race 4 (aka TropicalRace 4 or TR4) can be transformed into TR4-resistant plants bytransforming them with a nucleotide sequence coding for resistance usingthe banana transformation technologies provided in Example 4 and theFusR1 nucleotide sequences coding for TR4 resistance as provided herein.For example, a TR4-susceptible Cavendish banana cultivar can betransformed with one of the FusR1 alleles coding for TR4-resistance asprovided herein. As a further example, a TR4-susceptible Cavendishbanana cultivar can be transformed with one or more of the followingnucleotide coding sequences coding for TR4 resistance: SEQ ID NO: 2, SEQID NO: 5, SEQ ID NO: 9 SEQ ID NO: 11, SEQ ID NO:18, SEQ ID NO: 21,and/or SEQ ID NO:24.

For example, the Cavendish banana cultivar ‘Grand Nain’ (AAA) can betransformed with SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 9 and/or SEQ ID NO11 using the transformation protocols set forth in U.S. Pat. No.7,534,930 (‘Transgenic Disease Resistant Banana’), which is incorporatedherein in its entirety for everything it discloses.

In summary, immature male flowers of a Cavendish banana cultivar, such‘Grand Nain’ or ‘Williams,’ are used to produce embryogenic calli. Anucleic acid construct comprising SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:9, SEQ ID NO 11, SEQ ID NO:18, SEQ ID NO: 21, and/or SEQ ID NO:24,operably linked to a 35S promoter sequence is constructed. Or,alternatively, the promoter sequence of the FW-resistant allele 1 ofFusR1 from M. acuminata (SEQ ID NO 31) could be used to drive expressionof the resistance alleles. This construct is introduced into theembryogenic calli using microprojectile bombardment. Bombarded plantletsare regenerated from the embryogenic calli and the plantlets undergo PCRanalyses to determine which plantlets were transformed with theTR4-resistance gene(s). Tissue culture extracts from the resultingplants which positively express the TR4-resistance gene(s) are testedfor their ability to suppress growth of TR4. In addition, the putativetransformed plants are tested for resistance to TR4. TR4 resistantplants are isolated and cloned. The TR4 resistant plants can be used inbreeding programs to transfer the resistant genes as set forth inExample 3.

Where a transformed plant expresses SEQ ID NO 2 or SEQ ID NO 5; and,also expresses SEQ ID NO: 9 or SEQ ID NO: 11, that transformed plantwould have stacked resistance genes to TR4 given it comprises twodifferent nucleic acids coding for TR4 resistance. As discussed aboveand presented in Table 1, SEQ ID NO: 2 and SEQ ID NO: 5 are FusR1 allele1 and allele 2 coding sequences, respectively, coding for resistance asobtained from M. itinerans. In contrast, SEQ ID NO: 9 and SEQ ID NO: 11are FusR1 allele 1 and allele 2 coding sequences, respectively, codingfor resistance obtained from M. acuminata ssp. banksia. Thus, atransformed plant expressing both types of resistance genes would havestacked, or pyramidal, resistance to Panama Disease Tropical Race 4.

All resulting/selected banana plants with resistance to TR4 can bemaintained via asexual reproduction and used for production or insubsequent breeding programs.

Example 6: Banana Transformation Starting With a Cultivar ComprisingResistance

Transformed banana plants resistant to Panama Disease Tropical Race 4can be produced using the procedures outlined in Example 5 where theinitial, untransformed plant also has resistance to TR4 and/or to one ormore additional diseases. In this way the resultant transformed plantcan have multiple, or stacked, resistance genes. For example, thestarting cultivar used in the transformation procedures of Example 5 canbe a Cavenish cultivar with the resistance gene RGA2 (Dale et al.,2017). Thus, a Cavendish cultivar comprising the RGA2 coding sequencecan be transformed to express SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9and/or SEQ ID NO: 11, SEQ ID NO:18, SEQ ID NO: 21, and/or SEQ ID NO:24and thereby have stacked resistance genes to TR4.

All resulting/selected banana plants with resistance to TR4 can bemaintained via asexual reproduction and used for production or insubsequent breeding programs.

Example 7: Knocking Out Expression of FusR1-susceptibility Genes

In addition to or, alternatively instead of, transforming the plantsaccording to Example 5 or Example 6, the nucleotide sequences of FusR1alleles coding for susceptibility to TR4 in M acuminata (e.g., SEQ IDNO: 14) can be knocked-out using a TALEN, a meganuclease, a zinc fingernuclease, a CRISPR-associated nuclease or other appropriate gene editingtools.

In one such method, a guide RNA may be utilized along with anappropriate CRISPR-associated nuclease, including wherein the guide RNAcomprises a variable targeting domain that is complementary to all or apartial sequence of SEQ ID NO: 14. For example, a double-strand breakcan be introduced into an endogenous sequence coding for a FW-sensitiveFusR1 allele in M. acuminata (SEQ ID NO: 14) in a banana cell using amodified SEQ ID NO: 14, wherein the modified SEQ ID NO 14 comprises anucleic acid alteration that knocks out the gene function of SEQ ID NO:14.

For details on how to construct and use such a CRISPR-associatednuclease and Guide RNA in plants, see, for example, U.S. PatentApplication Publication No. 2019/0032070 A1 and WO 2019/118342 A1, eachof which is incorporated by reference in its entirety. For using CRISPRas a gene editing tool in banana, including to silence diseasesusceptibility genes, see, for example, WO 2018/220581 A1 (Compositionsand Methods for Increasing Shelf-Life of Banana); Tripahi et al., 2019,CRISPR/Cas9 editing of endogenous banana streak virus in the B genome ofMusa spp. overcomes a major challenge in banana breeding, CommunicationsBiology 2, Article 46, 11 pages; and, Ntui et al., January 2020, RobustCRISPR/Cas9 mediated genome editing tool for banana and plantain (Musaspp.), Vol. 21, 10 pages.

The modified plant cell can be generated/regenerated into a banana plantwhich can be maintained via asexual reproduction.

All resulting/selected banana plants with the knock out forsusceptibility to TR4 can be maintained via asexual reproduction andused for production or in subsequent breeding programs.

Example 8: Gene Editing of Bananas Susceptible to TR4

Banana plants susceptible to Fusarium oxysporum race 4 (aka TropicalRace 4 or TR4) can be modified into TR4-resistant plants by using genetargeting/gene editing tools to change their endogenous nucleic acidsequences coding for susceptibility into nucleotide sequences coding forresistance using the banana gene editing technologies provided inExample 4 and the FusR1 nucleotide sequences coding for TR4 resistanceas provided herein. For example, the endogenous nucleic acid sequencecoding for TR4-susceptibility in a Cavendish banana cultivar can bealtered based on the nucleic acid sequence of one of the FusR1 allelescoding for TR4-resistance as provided herein. As a further example, thenucleic acid sequence coding for TR4-susceptibility in a Cavendishbanana cultivar can be altered based on one or more of the followingnucleotide coding sequences coding for TR4 resistance: SEQ ID NO 2, SEQID NO 5, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO:18, SEQ ID NO: 21, and/orSEQ ID NO:24.

For example, the Cavendish banana cultivar ‘Grand Nain’ (AAA) can bemodified based on the nucleic acid sequences coding for resistance toTR4 as set forth herein (i.e., based upon SEQ ID NO 2, SEQ ID NO 5, SEQID NO 9, SEQ ID NO 11, SEQ ID NO:18, SEQ ID NO: 21, and/or SEQ ID NO:24)using modern gene editing tools. See FIG. 1.

In some general examples, the endogenous FW-susceptibility FusR1 gene ofSEQ ID NO 14 is modified by one or more of the following changes basedon its alignment with FW-resistant FusR1 genes of SEQ ID NO 2, SEQ ID NO5, SEQ ID NO, SEQ ID NO 11, SEQ ID NO:18, SEQ ID NO: 21, and/or SEQ IDNO:24. See FIG. 1.

In some specific examples, SEQ ID NO 14 is modified by the followingchanges based on its alignment with SEQ ID NO 9 (see sequence alignment,FIG. 1): the T corresponding to position 148 is replaced with G(148T>G); the T corresponding to position 323 is replaced with A(323T>A); the G corresponding to position 344 is replaced with C(344G>C); and/or, the A corresponding to position 347 is replaced with T(347A>T). In one example, the only substitution made is 344G>C. In oneexample, the following three substitutions are made: 323T>A, 344G>C and347A>T. In yet another example, all four substitutions are made: 148T>G,323T>A, 344G>C and 347A>T. See FIG. 1.

In some general examples, any and all nucleic acid substitutions aremade to the nucleic acid sequences coding for FW-susceptible FUSR1proteins so that the resulting, modified nucleic acids code forFW-resistant FUSR1 proteins. See FIG. 1 and FIG. 2.

In some specific examples, the endogenous nucleic acid sequence codingfor the FW-susceptible FUSR1 protein of SEQ ID NO: 15 is modified by oneor more nucleic acid changes based on its alignment with FW-resistantFUSR1 protein of SEQ ID NO: 12 to produce the following protein changes:the Leucine corresponding to position 50 is replaced with Valine(50L>V); the Valine corresponding to position 108 is replaced withGlutamic Acid (108V>E); the Arginine at position 115 is replaced withProline (115R>P); and/or, the Aspartic Acid at position 116 is replacedwith Valine (116D>V). In one example, the only protein substitution thatis made is 115R>P. In another example, the only protein substitutionsthat are made are 108V>E, 115R>P and 116D>V. In yet another example, allfour protein substitutions are made: 50L>V, 108V>E, 115R>P and 116D>V.See FIG. 2.

The banana-specific gene editing protocols from the followingpublications provide the protocols for making the necessary nucleotidebase pair substitutions in banana: Shao et al., 2020, Using CRISPR/Cas9genome editing system to create MaGA20ox2 gene-modified semi-dwarfbanana, Plant Biotechnology Journal, 18:17-19; Kaur et al., 2017,CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS)demonstrates precise manipulation in banana cv. Rasthali genome,Functional & Integrative Genomics, 18(1):89-99; Otang et al., 2020,Robust CRISPR/Cas9 mediated genome editing tool for banana and plantain(Musa spp.), Current Plant Biology, 21, 10 pages; Tripathi et al., 2019,CRISPR/Cas9 editing of endogenous banana streak virus in the B genome ofMusa spp. Overcomes a major challenge in banana breeding, CommunicationsBiology, 2:46, 11 pages; and, U.S. Pat. No. 7,381,556, each of which isentirely incorporated by reference herein for everything it teaches.

In summary, immature male flowers of a Cavendish banana cultivar, such‘Grand Nain’ or ‘Williams,’ is used to produce embryogenic calli and/oran embryogenic cell suspension. A CRISPR/Cas9 construct is preparedfollowing the procedures outline in any one or more of the above-listedscientific and patent publications, wherein the construct is constructedbased upon the sequence alignments provided in FIG. 1. The construct isdelivered into the embryogenic calli or embryogenic cell suspension andwell-rooted plantlets are generated. Random regenerates are selected andscreened for the presence of the Cas9 gene by PCR using primers. Thewell-rooted plantlets of Cas9 PCR-positive events and control plants areacclimatized and potted in the greenhouse. Molecular analyses areconducted to confirm gene editing in the endogenous FusR1 genes.

The genome edited plants and the control plants are evaluated foragronomic traits and evaluated for TR4 resistance. The resultinggene-edited plants which positively express the TR4-resistanceprotein(s) and display resistance to TR4 are cloned. The gene-edited TR4resistant plants can be used in breeding programs to transfer theresistant genes as set forth in Example 3.

All resulting/selected banana plants with resistance to TR4 can bemaintained via asexual reproduction and used for production or insubsequent breeding programs.

Further Numbered Embodiments of the Disclosure

Other subject matter contemplated by the present invention is set out inthe following numbered embodiments:

1. An isolated nucleic acid molecule comprising nucleic acid sequenceSEQ ID NO: 14 coding for susceptibility to Fusarium oxysporum race 4when expressed in a plant, wherein SEQ ID NO: 14 is modified by one,two, three or four nucleic acid substitutions so that the resultingnucleic acid sequence codes for resistance to Fusarium oxysporum race 4when expressed in a plant.2. The isolated nucleic acid molecule of embodiment 1, wherein thenucleic acid substitutions comprise replacing a T corresponding toposition 148 of SEQ ID NO: 14 with a G (148T>G).3. The isolated nucleic acid molecule of embodiment 1, wherein thenucleic acid substitutions comprise replacing a T corresponding toposition 323 of SEQ ID NO: 14 with an A (323T>A).4. The isolated nucleic acid molecule of embodiment 1, wherein thenucleic acid substitutions comprise replacing a G corresponding toposition 344 of SEQ ID NO: 14 with a C (344G>C).5. The isolated nucleic acid molecule of embodiment 1, wherein thenucleic acid substitutions comprise replacing an A corresponding toposition 347 of SEQ ID NO: 14 with a T (347A>T).6. The isolated nucleic acid molecule of embodiment 1, wherein thenucleic acid substitutions comprise replacing a T corresponding toposition 323 with an A (323T>A), replacing a G corresponding to position344 with a C (344G>C), and replacing an A corresponding to position 347with a T (347A>T), and wherein all positions are based on SEQ ID NO: 14.7. The isolated nucleic acid molecule of embodiment 1, wherein SEQ IDNO: 14 codes for an amino acid sequence of SEQ ID NO: 15 and wherein thenucleic acid substitutions result in replacing a Leucine correspondingto position 50 of SEQ ID NO: 15 with a Valine (50L>V).8. The isolated nucleic acid molecule of embodiment 1, wherein SEQ IDNO: 14 codes for an amino acid sequence of SEQ ID NO: 15 and wherein thenucleic acid substitutions result in replacing a Valine corresponding toposition 108 of SEQ ID NO: 15 with a Glutamic Acid (108V>E).9. The isolated nucleic acid molecule of embodiment 1, wherein SEQ IDNO: 14 codes for an amino acid sequence of SEQ ID NO: 15 and wherein thenucleic acid substitutions result in replacing an Arginine correspondingto position 115 of SEQ ID NO: 15 with a Proline (115R>P).10. The isolated nucleic acid molecule of embodiment 1, wherein SEQ IDNO: 14 codes for an amino acid sequence of SEQ ID NO: 15 and wherein thenucleic acid substitutions result in replacing an Aspartic Acidcorresponding to position 116 of SEQ ID NO: 15 with a Valine (116D>V).11. The isolated nucleic acid molecule of embodiment 1, wherein SEQ IDNO: 14 codes for an amino acid sequence of SEQ ID NO: 15 and wherein thenucleic acid substitutions result in replacing a Valine corresponding toposition 108 of SEQ ID NO: 15 with a Glutamic Acid (108V>E), an Argininecorresponding to position 115 of SEQ ID NO: 15 with a Proline (115R>P),and an Aspartic Acid corresponding to position 116 of SEQ ID NO: 15 witha Valine (116D>V).12. The isolated nucleic acid molecule of embodiments 1-11, wherein theexpression occurs in a plant cell, plant tissue, plant cell culture,plant tissue culture, or whole plant.13. The isolated nucleic acid molecule of embodiment 12, wherein theexpression occurs in aMusa cell, tissue, cell culture, tissue culture,or whole plant.14. The isolated nucleic acid molecule of embodiment 13, wherein theexpression occurs in aMusa acuminata cell, tissue, cell culture, tissueculture or whole plant.15. A nucleic acid construct comprising the isolated nucleic acidmolecule of embodiments 1-11, wherein the nucleic acid sequence isoperably linked to a promoter capable of driving expression of thenucleic acid sequence.16. The nucleic acid construct of embodiment 15, wherein the promoter isa plant promoter.17. The nucleic acid construct of embodiment 15, wherein the promoter isa 35S promoter.18. The nucleic acid construct of embodiment 15, wherein the promoter iscoded by SEQ ID NO: 31.19. A transformation vector comprising the nucleic acid construct ofembodiments 15-18.20. A method of transforming a plant cell comprising introducing thetransformation vector of embodiment 19 into a plant cell, whereby thetransformed plant cell expresses the nucleic acid sequence coding forresistance to Fusarium oxysporum race 4.21. The method of embodiment 20, wherein the plant cell is a Musa plantcell.22. The method of embodiment 20, wherein the plant cell is a Musaacuminata plant cell.23. The method of embodiments 20-22 further comprising producingtransformed plant tissue from the transformed plant cell.24. The method of embodiment 23 further comprising producing atransformed plantlet from the transformed plant tissue.25. The method of embodiment 24 further comprising producing a clone ofthe transformed plantlet.26. The method of embodiments 24 or 25 further comprising growing thetransformed plantlet or clone of the transformed plantlet into a maturetransformed plant.27. The method of embodiment 26, wherein the mature transformed plant isa Musa plant and the mature transformed Musa plant is capable ofproducing fruit.28. The method of embodiment 27 further comprising producing clones ofthe mature transformed Musa plant.29. The method of embodiment 27 or 28 further comprising using themature transformed Musa plant or clone of the mature transformed Musaplant in a breeding method.30. An isolated amino acid molecule comprising an amino acid sequence ofSEQ ID NO: 15 coding for a protein that when produced in a plant resultsin susceptibility to Fusarium oxysporum race 4, wherein SEQ ID NO: 15 ismodified by one, two, three or four amino acid substitutions so that itcodes for a protein which when produced in a plant results in resistanceto Fusarium oxysporum race 4.31. The isolated amino acid molecule of embodiment 30, wherein the aminoacid substitutions comprise replacing a Leucine corresponding toposition 50 of SEQ ID NO: 15 with a Valine (50L>V).32. The isolated amino acid molecule of embodiment 30, wherein the aminoacid substitutions comprise replacing a Valine corresponding to position108 of SEQ ID NO: 15 with a Glutamic Acid (108V>E)33. The isolated amino acid molecule of embodiment 30, wherein the aminoacid substitutions comprise replacing an Arginine corresponding toposition 115 of SEQ ID NO: 15 with a Proline (115R>P).34. The isolated amino acid molecule of embodiment 30, wherein the aminoacid substitutions comprise replacing an Aspartic Acid corresponding toposition 116 of SEQ ID NO: 15 with a Valine (116D>V).35. The isolated amino acid molecule of embodiment 30, wherein the aminoacid substitutions comprise replacing a Valine corresponding to position108 of SEQ ID NO: 15 with a Glutamic Acid (108V>E), an Argininecorresponding to position 115 of SEQ ID NO: 15 with a Proline (115R>P),and an Aspartic Acid corresponding to position 116 of SEQ ID NO: 15 witha Valine (116D>V).36. The isolated amino acid molecule segment of embodiments 30-35,wherein the production occurs in a plant cell, plant tissue, plant cellculture, plant tissue culture, or whole plant.37. The isolated amino acid molecule segment of embodiment 36, whereinthe production occurs in a Musa cell, tissue, cell culture, tissueculture, or whole plant.38. The isolated amino acid molecule segment of embodiment 36, whereinthe production occurs in a Musa acuminata cell, tissue, cell culture,tissue culture or whole plant.39. A nucleic acid construct comprising a nucleic acid sequence codingfor resistance to Fusarium oxysporum race 4 when expressed in a plant,wherein said nucleic acid sequence is selected from the group consistingof SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:18, SEQ ID NO: 21, and SEQ ID NO: 24, and wherein the nucleic acidsequence is operably linked to a promoter capable of driving expressionof the nucleic acid sequence.40. The nucleic acid construct of embodiment 39, wherein the promoter isa plant promoter.41. The nucleic acid construct of embodiment 39, wherein the promoter isa 35S promoter.42. The nucleic acid construct of embodiment 39, wherein the promoter iscoded by SEQ ID NO: 31.43. A transformation vector comprising the nucleic acid construct ofembodiments 39-42.44. A method of transforming a plant cell comprising introducing thetransformation vector of embodiment 43 into a plant cell, whereby thetransformed plant cell expresses the nucleic acid sequence coding forresistance to Fusarium oxysporum race 4.45. The method of embodiment 44, wherein the plant cell is a Musa plantcell.46. The method of embodiment 44, wherein the plant cell is a Musaacuminata plant cell.47. The method of embodiments 44-46 further comprising producingtransformed plant tissue from the transformed plant cell.48. The method of embodiment 47 further comprising producing atransformed plantlet from the transformed plant tissue.49. The method of embodiment 48 further comprising producing a clone ofthe transformed plantlet.50. The method of embodiments 48 or 49 further comprising growing thetransformed plantlet or clone of the transformed plantlet into a maturetransformed plant.51. The method of embodiment 50, wherein the mature transformed plant isa Musa plant and the mature transformed Musa plant is capable ofproducing fruit.52. The method of embodiment 51 further comprising producing clones ofthe mature transformed Musa plant.53. The method of embodiments 51 or 52 further comprising using themature transformed Musa plant or clone of the mature transformed Musaplant in a breeding method.54. A banana breeding method comprising crossing a first Musa plantcomprising a nucleic acid sequence coding for resistance to Fusariumoxysporum race 4 with a second Musa plant that is susceptible toFusarium oxysporum race 4 and selecting resultant progeny of the crossbased on their resistance to Fusarium oxysporum race 4, wherein saidnucleic acid sequence coding for resistance to Fusarium oxysporum race 4is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO:24.55. The banana breeding method of embodiment 54 further comprisingproducing clones of the resultant progeny of the cross wherein theclones are selected based on their resistance to Fusarium oxysporum race4.56. The banana breeding method of embodiment 54, wherein the first andsecond Musa plants are from different Musa species. The banana breedingmethod of embodiment 54, wherein the first and second Musa plants arefrom the same Musa species. The banana breeding method of embodiment 54,wherein the first and/or second Musa plant is a Musa acuminata plant.57. The banana breeding method of embodiment 54, wherein the progeny ofthe cross that display resistance to Fusarium oxysporum race 4 areselected using molecular markers that are designed based on the nucleicacid sequence coding for resistance to Fusarium oxysporum race 4 that ispresent in the first Musa plant used in the cross.58. A method for obtaining a Musa acuminata plant cell with a silencedendogenous gene coding for susceptibility to Fusarium oxysporum race 4,the method comprising introducing a double-strand break to at least onesite in an exogenous gene coded by SEQ ID NO: 14 to produce a Musaacuminata plant cell with a silenced endogenous gene coding forsusceptibility to Fusarium oxysporum race 4.59. The method of embodiment 58 further comprising generating a Musaacuminata plant from the Musa acuminata plant cell with a silencedendogenous gene coding for susceptibility to Fusarium oxysporum race 4to produce a Musa acuminata plant with a silenced endogenous gene codingfor susceptibility to Fusarium oxysporum race 4.60. The method of embodiment 59 further comprising using the Musaacuminata plant with a silenced endogenous gene coding forsusceptibility to Fusarium oxysporum race 4 in a banana breedingprogram.61. The method of embodiment 20 or 44, wherein the plant cell is theMusa acuminata plant cell of embodiment 59 with a silenced endogenousgene coding for susceptibility to Fusarium oxysporum race 4.62. The method of embodiment 58, wherein the double-strand break isinduced by a nuclease selected from the group consisting of a TALEN, ameganuclease, a zinc finger nuclease, and a CRISPR-associated nuclease.63. The method of claim 62, wherein the double-strand break is inducedby a CRISPR-associated nuclease and where a guide RNA is provided.64. A method for producing a plant cell resistant to Fusarium oxysporumrace 4 comprising introducing at least one genetic modification into oneor more endogenous nucleic acid sequences coding for susceptibility toFusarium oxysporum race 4, wherein the genetic modification confersresistance to Fusarium oxysporum race 4 to the plant cell.65. The method of embodiment 64 wherein the at least one geneticmodification is introduced by a TALEN, a meganuclease, a zinc fingernuclease or a CRISPR-associated nuclease.66. The method of claim 64, wherein the at least one geneticmodification is introduced by a CRISPR-associated nuclease and anassociated guide RNA.67. The method of embodiment 64, wherein the at least one geneticmodification is selected from the list consisting of replacing a Tcorresponding to position 148 of SEQ ID NO: 14 with a G (148T>G),replacing a T corresponding to position 323 of SEQ ID NO: 14 with an A(323T>A), replacing a G corresponding to position 344 of SEQ ID NO: 14with a C (344G>C), and replacing an A corresponding to position 347 ofSEQ ID NO: 14 with a T (347A>T).68. The method of embodiment 64, wherein the at least one geneticmodification results in a change in an amino acid selected from thegroup consisting of replacing a Leucine corresponding to position 50 ofSEQ ID NO: 15 with a Valine (50L>V), replacing a Valine corresponding toposition 108 of SEQ ID NO: 15 with a Glutamic Acid (108V>E), replacingan Arginine corresponding to position 115 of SEQ ID NO: 15 with aProline (115R>P), and replacing an Aspartic Acid corresponding toposition 116 of SEQ ID NO: 15 with a Valine (116D>V).69. The method of embodiments 64-68, wherein the plant cell is aMusaplant cell.70. The method of embodiments 64-68, wherein the plant cell is aMusaacuminata plant cell.71. The method of embodiments 64-70 further comprising producingtransformed plant tissue from the transformed plant cell.72. The method of embodiment 71 further comprising producing atransformed plantlet from the transformed plant tissue.73. The method of embodiment 72 further comprising producing a clone ofthe transformed plantlet.74. The method of embodiments 71 or 72 further comprising growing thetransformed plantlet or clone of the transformed plantlet into a maturetransformed plant.75. The method of embodiment 74, wherein the mature transformed plant isa Musa plant and the mature transformed Musa plant is capable ofproducing fruit.76. The method of embodiment 75 further comprising producing clones ofthe mature transformed Musa plant.77. The method of embodiments 75 or 76 further comprising using themature transformed Musa plant or clone of the mature transformed Musaplant in a breeding method.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein within the above text and/or citedbelow are incorporated by reference in their entireties for allpurposes. However, mention of any reference, article, publication,patent, patent publication, and patent application cited herein is not,and should not be taken as acknowledgment or any form of suggestion thatthey constitute valid prior art or form part of the common generalknowledge in any country in the world.

U. S. PATENT DOCUMENTS

-   U.S. Pat. No. 7,534,930 B2 May 2009 Vishnevetsky et al.-   U.S. Pat. No. 6,274,319 August 2001 Messier and Sikela-   U.S. Pat. No. 9,834,783 December 2017 Messier

OTHER PUBLICATIONS

-   Armenteros, J. J. A. A. 2017. DeepLoc: prediction of protein    subcellular localization using deep learning. Bioinformatics 33(21):    3387-3395.-   Armenteros et al. 2019. SignalP 5.0 improves signal peptide    predictions using deep neural networks. Nat Biotechnol 37:420-423.-   Bai, T-T. et al. 2013. Transcriptome and Expression Profile Analysis    of Highly Resistant and Susceptible Banana Roots Challenged with    Fusarium oxysporum f sp. cubense Tropical Race 4. PLOS|One    Published: Sep. 23, 2013.-   Barbosa, J. A. R. G. et al., 2007. Crystal Structure of the    Bowman-Birk Inhibitor from Vigna unguiculata Seeds in Complex with    β-Trypsin at 1.55 A Resolution and Its Structural Properties in    Association with Proteinases. Biophysical Journal. 92(5): 1638-1650.-   Chen, A., et al. 2019. Assessing Variations in Host Resistance to    Fusarium oxysporum f sp. cubense Race 4 in Musa Species, With a    Focus on the Subtropical Race 4. Front. Microbiol. 10.-   Christelova, P. et al. 2017. Molecular and cytological    characterization of the global Musa germplasm collection provides    insights into the treasure of banana diversity. Biodivers. Conserv.    26: 801.-   Dale, J. et al. 2017. Transgenic Cavendish bananas with resistance    to Fusarium wilt tropical race 4. Nature Communications. 8: Article    number 1496.-   Davey, M. W. et al. 2013. A draft Musa balbisiana genome sequence    for molecular genetics in polyploid, inter- and intra-specific Musa    hybrids. BMC Genomics 14: 683.-   D'Hont, A. et al. 2012. The banana (Musa acuminata) genome and the    evolution of monocotyledonous plants. Nature 488:213-217.-   Dita, M. et al. 2018. Fusarium Wilt of banana: current knowledge on    epidemiology and research needs toward sustainable disease    management. Front Plant Sci. 9:1468.-   Heslop-Harrison, J. S. and Schwarzacher, T. 2007. Domestication,    Genomics and the Future for Banana. Annals of Botany    100(5):1073-1084.-   Hiller, K, et al. 2004. PrediSi: prediction of signal peptides and    their cleavage positions. Nucleic Acids Res. 32(Web Server    issue):W375-9.-   Hippolyte, I. et al. 2012. Foundation characteristics of edible Musa    triploids revealed from allelic distribution of SSR markers. Annals    of Botany 109(5):937-951.-   Hughes, A. L and Nei, M. 1988 Nature 335:167-170.-   Ishihara et al. 2016. An improved method for RNA extraction from    woody legume species Acacia koa A. Gray and Leucaena leucocephala    (Lam.) de Wit. Int. J. For. Wood Sci. 3(1): 31-35.-   Kreitman, M. and Akashi, H. 1995. Molecular evidence for natural    selection. Annu. Rev. Ecol. Syst. 26:403-422.-   Kumar, S., et al. 2018. MEGA X: Molecular Evolutionary Genetics    Analysis across computing platforms. Molecular Biology and Evolution    35:1547-1549.-   Li, C.-Y. et al. 2012. Transcriptome profiling of resistant and    susceptible Cavendish banana roots following inoculation with    Fusarium oxysporum f. sp. cubense tropical race 4. BMC Genomics    13:374.-   Li, W.-H. et al. 1985. A new method for estimating synonymous and    nonsynonymous rates of nucleotide substitution considering the    relative likelihood of nucleotide and codon changes. Mol. Biol.    Evol. 2: 150-174.-   Li, W.-H. 1993. Unbiased estimation of the rates of synonymous and    nonsynonymous substitution. J. Mol. Evol. 36: 9699.-   Li, W.-H., 1997. Molecular Evolution. Sunderland, Mass.: Sinauer    Associates.-   Li, W. M. et al. 2015. Resistance sources to Fusarium oxysporum f    sp. cubense tropical race 4 in banana wild relatives. Plant    Pathology 64:1061-1067.-   Ma, X, et al. 2015 A Robust CRISPR/Cas9 System for Convenient,    High-Efficiency Multiplex Genome Editing in Monocot and Dicot    Plants. Mol. Plant. 8:1274-1284.-   Messier, W. and Stewart, C.-B. 1994 Current Biol. 4:911-913.-   Messier, W. and Stewart, C-B. 1997. Nature 385:151-154.-   Nei M. and Kumar S. 2000. Molecular Evolution and Phylogenetics.    Oxford University Press, New York.-   Paul, J.-Y. et al. 2011. Apoptosis-related genes confer resistance    to Fusarium wilt in transgenic ‘Lady Finger’ bananas. Plant    Biotechnology Journal.-   Niu, Y. et al. 2018. Comparative digital gene expression analysis of    tissue-cultured plantlets of highly resistant and susceptible banana    cultivars in response to Fusarium oxysporum. Int. J. Mol. Sci. 19.    doi: 10.3390/ijms19020350.-   Peraza-Echeverria, S. et al. 2009. Molecular cloning and in silico    analysis of potential Fusarium resistance genes in banana. Mol.    Breeding. 23(3): 431-443.-   Ploetz, R. C. 2015. Fusarium Wilt of banana. Phytopathology Review.-   Raboin, L-M. et al. 2005. Diploid Ancestors of Triploid Export    Banana Cultivars: Molecular Identification of 2n Restitution Gamete    Donors and n Gamete Donors. Mol Breeding 16:333.-   Reese M. G. 2001. Application of a time-delay neural network to    promoter annotation in the Drosophila melanogaster genome. Comput.    Chem. 26(1): 51-56.-   Ribeiro, L. R. et al. 2018. Sources of resistance to Fusarium    oxysporum f sp. cubense in banana germplasm. Rev. Bras. Frutic.    40:1. Epub Feb. 8, 2018.-   Rouard, M. et al. 2018. Three New Genome Assemblies Support a Rapid    Radiation in Musa acuminata (Wild Banana). Genome Biology and    Evolution 10(12): 3129-3140.-   Solovyev V. V. and Salamov A. A. 1997. The Gene-Finder computer    tools for analysis of human and model organisms genome sequences. In    Proceedings of the Fifth International Conference on Intelligent    Systems for Molecular Biology (eds. Rawling C., Clark D., Altman R.,    Hunter L., Lengauer T., Wodak S.), Halkidiki, Greece, AAAI Press,    294-302.-   Solovyev V. V. 2001. Statistical approaches in Eukaryotic gene    prediction. In Handbook of Statistical Genetics (eds. Balding D. et    al.), John Wiley & Sons, Ltd., p. 83-127.-   Solovyev V. V. and Shahmuradov I. A. 2003. PromH: Promoters    identification using orthologous genomic sequences. Nucleic Acids    Res. 31(13): 3540-3545.-   Stokstad, E. 2019. Banana fungus puts Latin America on alert.    Science 365(6450): 207-208.-   Ssali, R. et al. 2013. Inheritance of resistance to Fusarium    oxysporum f sp. cubense race 1 in bananas. Euphytica 194: 425. Van    der Berg, N. et al. 2007. Tolerance in banana to Fusarium wilt is    associated with early up-regulation of cell wall-strengthening genes    in the roots. Molecular Plant Pathology. 8(3): 333-341.-   Venkataramana, R. K. et al. 2015. Insights into Musa balbisiana and    Musa acuminata species divergence and development of genic    microsatellites by transcriptomics approach. Plant Gene 4: 78-82.-   Wang, Y. et al. 2017. Differential gene expression in banana roots    in response to Fusarium wilt. Canadian Journal of Plant Pathology    39(2): 163-175. doi.org/10.1080/07060661.2017.1342693.-   Wu, W. et al. 2016. Whole genome sequencing of a banana wild    relative Musa itinerans provides insights into lineage-specific    diversification of the Musa genus. Scientific Reports 6: Article    number: 31586.-   Zhang, L. et al. (2018) Identification and evaluation of resistance    to Fusarium oxysporum f. sp. cubense tropical race 4 in Musa    acuminata Pahang. Euphytica 214: 106.-   Zuo, C. et al. 2018. Germplasm screening of Musa spp. for resistance    to Fusarium oxysporum f sp. cubense tropical race 4 (Foc-TR4). Eur J    Plant Pathol. 151:723.

1.-38. (canceled)
 39. A nucleic acid construct comprising a nucleic acidsequence coding for resistance to Fusarium oxysporum race 4 whenexpressed in a plant, wherein said nucleic acid sequence is selectedfrom the group consisting of a nucleic acid sequence having at least 95%a sequence identity to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 24, and wherein thenucleic acid sequence is operably linked to a promoter capable ofdriving expression of the nucleic acid sequence.
 40. The nucleic acidconstruct of claim 39, wherein the promoter is a plant promoter.
 41. Thenucleic acid construct of claim 39, wherein the promoter is a 35Spromoter.
 42. The nucleic acid construct of claim 39, wherein thepromoter is coded by SEQ ID NO:
 31. 43. A transgenic plant, plant part,plant cell, or a plant tissue culture comprising a nucleic acidconstruct comprising a nucleic acid sequence coding for resistance toFusarium oxysporum race 4 when expressed in a plant, wherein saidnucleic acid sequence is selected from the group consisting of a NO: 5,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ IDNo: 24, and wherein the nucleic acid sequence is operably linked to apromoter capable of driving expression of the nucleic acid sequence. 44.A method of transforming a plant cell comprising introducing the nucleicacid construct of claim 39 into a plant cell, whereby the transformedplant cell expresses the nucleic acid sequence coding for resistance toFusarium oxysporum race
 4. 45. The method of claim 44, wherein the plantcell is a Musa plant cell.
 46. The method of claim 44, wherein the plantcell is a Musa acuminata plant cell.
 47. The method of claim 44, furthercomprising producing a transformed plant tissue from the transformedplant cell.
 48. The method of claim 47, further comprising producing atransformed plantlet from the transformed plant tissue.
 49. The methodof claim 48, further comprising producing a clone of the transformedplantlet.
 50. The method of claim 48, further comprising growing thetransformed plantlet of the transformed plantlet into a maturetransformed plant.
 51. The method of claim 50, wherein the maturetransformed plant is a Musa plant and the mature transformed Musa plantis capable of producing fruit.
 52. The method of claim 51, furthercomprising producing clones of the mature transformed Musa plant. 53.The method of claim 51, further comprising using the mature transformedMusa plant of the mature transformed Musa plant in a breeding method.54.-77. (canceled)
 78. The transgenic plant of claim 43, wherein thepromoter is a plant promoter.
 79. The transgenic plant of claim 43,wherein the promoter is a 35S promoter.
 80. The transgenic plant ofclaim 43, wherein the promoter is coded by SEQ ID NO: 31.